Nucleic acid sequences to proteins involved in tocopherol synthesis

ABSTRACT

Nucleic acid sequences and methods are provided for producing plants and seeds having altered tocopherol content and compositions. The methods find particular use in increasing the tocopherol levels in plants, and in providing desirable tocopherol compositions in a host plant cell.

TECHNICAL FIELD

The present invention is directed to nucleic acid and amino acidsequences and constructs, and methods related thereto.

BACKGROUND

Isoprenoids are ubiquitous compounds found in all living organisms.Plants synthesize a diverse array of greater than 22,000 isoprenoids(Connolly and Hill (1992) Dictionary of Terpenoids, Chapman and Hall,New York, N.Y.). In plants, isoprenoids play essential roles inparticular cell functions such as production of sterols, contributing toeukaryotic membrane architecture, acyclic polyprenoids found in the sidechain of ubiquinone and plastoquinone, growth regulators like abscisicacid, gibberellins, brassinosteroids or the photosynthetic pigmentschlorophylls and carotenoids. Although the physiological role of otherplant isoprenoids is less evident, like that of the vast array ofsecondary metabolites, some are known to play key roles mediating theadaptative responses to different environmental challenges. In spite ofthe remarkable diversity of structure and function, all isoprenoidsoriginate from a single metabolic precursor, isopentenyl diphosphate(IPP) (Wright, (1961) Annu. Rev. Biochem. 20:525-548; and Spurgeon andPorter, (1981) in Biosynthesis of Isoprenoid Compounds., Porter andSpurgeon eds (John Wiley, New York) Vol. 1, pp 1-46).

A number of unique and interconnected biochemical pathways derived fromthe isoprenoid pathway leading to secondary metabolites, includingtocopherols, exist in chloroplasts of higher plants. Tocopherols notonly perform vital functions in plants, but are also important frommammalian nutritional perspectives. In plastids, tocopherols account forup to 40% of the total quinone pool.

Tocopherols and tocotrienols (unsaturated tocopherol derivatives) arewell known antioxidants, and play an important role in protecting cellsfrom free radical damage, and in the prevention of many diseases,including cardiac disease, cancer, cataracts, retinopathy, Alzheimer'sdisease, and neurodegeneration, and have been shown to have beneficialeffects on symptoms of arthritis, and in anti-aging. Vitamin E is usedin chicken feed for improving the shelf life, appearance, flavor, andoxidative stability of meat, and to transfer tocols from feed to eggs.Vitamin E has been shown to be essential for normal reproduction,improves overall performance, and enhances immunocompetence in livestockanimals. Vitamin E supplement in animal feed also imparts oxidativestability to milk products.

The demand for natural tocopherols as supplements has been steadilygrowing at a rate of 10-20% for the past three years. At present, thedemand exceeds the supply for natural tocopherols, which are known to bemore biopotent than racemic mixtures of synthetically producedtocopherols. Naturally occurring tocopherols are all d-stereomers,whereas synthetic α-tocopherol is a mixture of eight d,l-α-tocopherolisomers, only one of which (12.5%) is identical to the naturald-α-tocopherol. Natural d-α-tocopherol has the highest vitamin Eactivity (1.49 IU/mg) when compared to other natural tocopherols ortocotrienols. The synthetic α-tocopherol has a vitamin E activity of 1.1IU/mg. In 1995, the worldwide market for raw refined tocopherols was$1020 million; synthetic materials comprised 85-88% of the market, theremaining 12-15% being natural materials. The best sources of naturaltocopherols and tocotrienols are vegetable oils and grain products.Currently, most of the natural Vitamin E is produced from γ-tocopherolderived from soy oil processing, which is subsequently converted toα-tocopherol by chemical modification (α-tocopherol exhibits thegreatest biological activity).

Methods of enhancing the levels of tocopherols and tocotrienols inplants, especially levels of the more desirable compounds that can beused directly, without chemical modification, would be useful to the artas such molecules exhibit better functionality and biovailability.

In addition, methods for the increased production of other isoprenoidderived compounds in a host plant cell is desirable. Furthermore,methods for the production of particular isoprenoid compounds in a hostplant cell is also needed.

SUMMARY OF THE INVENTION

The present invention is directed to sequences to proteins involved intocopherol synthesis. The polynucleotides and polypeptides of thepresent invention include those derived from prokaryotic and eukaryoticsources.

Thus, one aspect of the present invention relates to prenyltransferase,and in particular to isolated polynucleotide sequences encodingprenyltransferase proteins and polypeptides related thereto. Inparticular, isolated nucleic acid sequences encoding prenyltransferaseproteins from bacterial and plant sources are provided.

In another aspect, the present invention provides isolatedpolynucleotide sequences encoding tocopherol cyclase, and polypeptidesrelated thereto. In particular, isolated nucleic acid sequences encodingtocopherol cyclase proteins from bacterial and plant sources areprovided.

Another aspect of the present invention relates to oligonucleotideswhich include partial or complete prenyltransferase or tocopherolcyclase encoding sequences.

It is also an aspect of the present invention to provide recombinant DNAconstructs which can be used for transcription or transcription andtranslation (expression) of prenyltransferase or tocopherol cyclase. Inparticular, constructs are provided which are capable of transcriptionor transcription and translation in host cells.

In another aspect of the present invention, methods are provided forproduction of prenyltransferase or tocopherol cyclase in a host cell orprogeny thereof. In particular, host cells are transformed ortransfected with a DNA construct which can be used for transcription ortranscription and translation of prenyltransferase or tocopherolcyclase. The recombinant cells which contain prenyltransferase ortocopherol cyclase are also part of the present invention.

In a further aspect, the present invention relates to methods of usingpolynucleotide and polypeptide sequences to modify the tocopherolcontent of host cells, particularly in host plant cells. Plant cellshaving such a modified tocopherol content are also contemplated herein.Methods and cells in which both prenyltransferase and tocopherol cyclaseare expressed in a host cell are also part of the present invention.

The modified plants, seeds and oils obtained by the expression of theprenyltransferase or tocopherol cyclase are also considered part of theinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides an amino acid sequence alignment between ATPT2, ATPT3,ATPT4, ATPT8, and ATPT12 are performed using ClustalW.

FIG. 2 provides a schematic picture of the expression constructpCGN10800.

FIG. 3 provides a schematic picture of the expression constructpCGN10801.

FIG. 4 provides a schematic picture of the expression constructpCGN10803.

FIG. 5 provides a schematic picture of the construct pCGN10806.

FIG. 6 provides a schematic picture of the construct pCGN10807.

FIG. 7 provides a schematic picture of the construct pCGN10808.

FIG. 8 provides a schematic picture of the expression constructpCGN10809.

FIG. 9 provides a schematic picture of the expression constructpCGN10810.

FIG. 10 provides a schematic picture of the expression constructpCGN10811.

FIG. 11 provides a schematic picture of the expression constructpCGN10812.

FIG. 12 provides a schematic picture of the expression constructpCGN10813.

FIG. 13 provides a schematic picture of the expression constructpCGN10814.

FIG. 14 provides a schematic picture of the expression constructpCGN10815.

FIG. 15 provides a schematic picture of the expression constructpCGN10816.

FIG. 16 provides a schematic picture of the expression constructpCGN10817.

FIG. 17 provides a schematic picture of the expression constructpCGN10819.

FIG. 18 provides a schematic picture of the expression constructpCGN10824.

FIG. 19 provides a schematic picture of the expression constructpCGN10825.

FIG. 20 provides a schematic picture of the expression constructpCGN10826.

FIG. 21 provides an amino acid sequence alignment using ClustalW betweenthe Synechocystis prenyltransferase sequences.

FIG. 22 provides an amino acid sequence of the ATPT2, ATPT3, ATPT4,ATPT8, and ATPT12 protein sequences from Arabidopsis and the slr1736,slr0926, sll1899, slr0056, and the slr1518 amino acid sequences fromSynechocystis.

FIG. 23 provides the results of the enzymatic assay from preparations ofwild type Synechocystis strain 6803, and Synechocystis slr1736 knockout.

FIG. 24 provides bar graphs of HPLC data obtained from seed extracts oftransgenic Arabidopsis containing pCGN10822, which provides of theexpression of the ATPT2 sequence, in the sense orientation, from thenapin promoter. Provided are graphs for alpha, gamma, and deltatocopherols, as well as total tocopherol for 22 transformed lines, aswell as a nontransformed (wildtype) control.

FIG. 25 provides a bar graph of HPLC analysis of seed extracts fromArabidopsis plants transformed with pCGN10803 (³⁵S-ATPT2, in theantisense orientation), pCGN10822 (line 1625, napin ATPT2 in the senseorientation), pCGN10809 (line 1627, 35S-ATPT3 in the sense orientation),a nontransformed (wt) control, and an empty vector transformed control.

FIG. 26 shows total tocopherol levels measured in T# Arabidopsis seed ofline.

FIG. 27 shows total tocopherol levels measured in T# Arabidopsis seed ofline.

FIG. 28 shows total tocopherol levels measured in developing canola seedof line 10822-1.

FIG. 29: shows results of phytyl prenyltransferase activity assay usingSynechocystis wild type and slr1737 knockout mutant membranepreparations.

FIG. 30 is the chromatograph from an HPLC analysis of Synechocystisextracts.

FIG. 31 is a sequence alignment of the Arabidopsis homologue with thesequence of the public database.

FIG. 32 shows the results of hydropathic analysis of slr1737

FIG. 33 shows the results of hydropathic analysis of the Arabidopsishomologue of ski 737.

FIG. 34 shows the catalytic mechanism of various cyclase enzymes

FIG. 35 is a sequence alignment of slr1737, slr1737 Arabidopsishomologue and the Arabidopsis chalcone isomerase.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides, inter alia, compositions and methods foraltering (for example, increasing and decreasing) the tocopherol levelsand/or modulating their ratios in host cells. In particular, the presentinvention provides polynucleotides, polypeptides, and methods of usethereof for the modulation of tocopherol content in host plant cells.

The biosynthesis of α-tocopherol in higher plants involves condensationof homogentisic acid and phytylpyrophosphate to form 2-methyl-6phytylbenzoquinol that can, by cyclization and subsequent methylations(Fiedler et al., 1982, Planta, 155: 511-515, Soll et al., 1980, Arch.Biochem. Biophys. 204: 544-550, Marshall et al., 1985 Phytochem., 24:1705-1711, all of which are herein incorporated by reference in theirentirety), form various tocopherols.

The Arabidopsis pds2 mutant identified and characterized by Norris etal. (1995), is deficient in tocopherol and plastiquinone-9 accumulation.Further genetic and biochemical analysis suggested that the proteinencoded by PDS2 may be responsible for the prenylation of homogentisicacid. The PDS2 locus identified by Norris et al. (1995) has beenhypothesized to possibly encode the tocopherol phytyl-prenyltransferase,as the pds2 mutant fails to accumulate tocopherols.

Norris et al. (1995) determined that in Arabidopsis pds2 lies at the topof chromosome 3, approximately 7 centimorgans above long hypocotyl2,based on the genetic map. ATPT2 is located on chromosome 2 between 36and 41 centimorgans, lying on BAC F19F24, indicating that ATPT2 does notcorrespond to PDS2. Thus, it is an aspect of the present invention toprovide novel polynucleotides and polypeptides involved in theprenylation of homogentisic acid. This reaction may be a rate limitingstep in tocopherol biosynthesis, and this gene has yet to be isolated.

U.S. Pat. No. 5,432,069 describes the partial purification andcharacterization of tocopherol cyclase from Chlorella protothecoides,Dunaliella salina and wheat. The cyclase described as being glycinerich, water soluble and with a predicted MW of 48-50 kDa. However, onlylimited peptide fragment sequences were available.

In one aspect, the present invention provides polynucleotide andpolypeptide sequences involved in the prenylation of straight chain andaromatic compounds. Straight chain prenyltransferases as used hereincomprises sequences which encode proteins involved in the prenylation ofstraight chain compounds, including, but not limited to, geranyl geranylpyrophosphate and farnesyl pyrophosphate. Aromatic prenyltransferases,as used herein, comprises sequences which encode proteins involved inthe prenylation of aromatic compounds, including, but not limited to,menaquinone, ubiquinone, chlorophyll, and homogentisic acid. Theprenyltransferase of the present invention preferably prenylateshomogentisic acid.

In another aspect, the invention provides polynucleotide and polypeptidesequences to tocopherol cyclization enzymes. The2,3-dimethyl-5-phytylplastoquinol cyclase (tocopherol cyclase) isresponsible for the cyclization of 2,3-dimethyl-5-phytylplastoquinol totocopherol.

Isolated Polynucleotides Proteins, and Polypeptides

A first aspect of the present invention relates to isolatedprenyltransferase polynucleotides. Another aspect of the presentinvention relates to isolated tocopherol cyclase polynucleotides. Thepolynucleotide sequences of the present invention include isolatedpolynucleotides that encode the polypeptides of the invention having adeduced amino acid sequence selected from the group of sequences setforth in the Sequence Listing and to other polynucleotide sequencesclosely related to such sequences and variants thereof.

The invention provides a polynucleotide sequence identical over itsentire length to each coding sequence as set forth in the SequenceListing. The invention also provides the coding sequence for the maturepolypeptide or a fragment thereof, as well as the coding sequence forthe mature polypeptide or a fragment thereof in a reading frame withother coding sequences, such as those encoding a leader or secretorysequence, a pre-, pro-, or prepro-protein sequence. The polynucleotidecan also include non-coding sequences, including for example, but notlimited to, non-coding 5′ and 3′ sequences, such as the transcribed,untranslated sequences, termination signals, ribosome binding sites,sequences that stabilize mRNA, introns, polyadenylation signals, andadditional coding sequence that encodes additional amino acids. Forexample, a marker sequence can be included to facilitate thepurification of the fused polypeptide. Polynucleotides of the presentinvention also include polynucleotides comprising a structural gene andthe naturally associated sequences that control gene expression.

The invention also includes polynucleotides of the formula:

X—(R₁)_(n)—(R₂)—(R₃)_(n)—Y

wherein, at the 5′ end, X is hydrogen, and at the 3′ end, Y is hydrogenor a metal, R₁ and R₃ are any nucleic acid residue, n is an integerbetween 1 and 3000, preferably between 1 and 1000 and R₂ is a nucleicacid sequence of the invention, particularly a nucleic acid sequenceselected from the group set forth in the Sequence Listing and preferablythose of SEQ ID NOs: 1, 3, 5, 7, 8, 10, 11, 13-16, 18, 23, 29, 36, and38. In the formula, R₂ is oriented so that its 5′ end residue is at theleft, bound to R₁, and its 3′ end residue is at the right, bound to R₃.Any stretch of nucleic acid residues denoted by either R group, where Ris greater than 1, may be either a heteropolymer or a homopolymer,preferably a heteropolymer.

The invention also relates to variants of the polynucleotides describedherein that encode for variants of the polypeptides of the invention.Variants that are fragments of the polynucleotides of the invention canbe used to synthesize full-length polynucleotides of the invention.Preferred embodiments are polynucleotides encoding polypeptide variantswherein 5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid residues of apolypeptide sequence of the invention are substituted, added or deleted,in any combination. Particularly preferred are substitutions, additions,and deletions that are silent such that they do not alter the propertiesor activities of the polynucleotide or polypeptide.

Further preferred embodiments of the invention that are at least 50%,60%, or 70% identical over their entire length to a polynucleotideencoding a polypeptide of the invention, and polynucleotides that arecomplementary to such polynucleotides. More preferable arepolynucleotides that comprise a region that is at least 80% identicalover its entire length to a polynucleotide encoding a polypeptide of theinvention and polynucleotides that are complementary thereto. In thisregard, polynucleotides at least 90% identical over their entire lengthare particularly preferred, those at least 95% identical are especiallypreferred. Further, those with at least 97% identity are highlypreferred and those with at least 98% and 99% identity are particularlyhighly preferred, with those at least 99% being the most highlypreferred.

Preferred embodiments are polynucleotides that encode polypeptides thatretain substantially the same biological function or activity as themature polypeptides encoded by the polynucleotides set forth in theSequence Listing.

The invention further relates to polynucleotides that hybridize to theabove-described sequences. In particular, the invention relates topolynucleotides that hybridize under stringent conditions to theabove-described polynucleotides. As used herein, the terms “stringentconditions” and “stringent hybridization conditions” mean thathybridization will generally occur if there is at least 95% andpreferably at least 97% identity between the sequences. An example ofstringent hybridization conditions is overnight incubation at 42° C. ina solution comprising 50% formamide, 5×SSC (150 mM NaCl, 15 mM trisodiumcitrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10%dextran sulfate, and 20 micrograms/milliliter denatured, sheared salmonsperm DNA, followed by washing the hybridization support in 0.1×SSC atapproximately 65° C. Other hybridization and wash conditions are wellknown and are exemplified in Sambrook, et al., Molecular Cloning: ALaboratory Manual, Second Edition, cold Spring Harbor, N.Y. (1989),particularly Chapter 11.

The invention also provides a polynucleotide consisting essentially of apolynucleotide sequence obtainable by screening an appropriate librarycontaining the complete gene for a polynucleotide sequence set for inthe Sequence Listing under stringent hybridization conditions with aprobe having the sequence of said polynucleotide sequence or a fragmentthereof; and isolating said polynucleotide sequence. Fragments usefulfor obtaining such a polynucleotide include, for example, probes andprimers as described herein.

As discussed herein regarding polynucleotide assays of the invention,for example, polynucleotides of the invention can be used as ahybridization probe for RNA, cDNA, or genomic DNA to isolate full lengthcDNAs or genomic clones encoding a polypeptide and to isolate cDNA orgenomic clones of other genes that have a high sequence similarity to apolynucleotide set forth in the Sequence Listing. Such probes willgenerally comprise at least 15 bases. Preferably such probes will haveat least 30 bases and can have at least 50 bases. Particularly preferredprobes will have between 30 bases and 50 bases, inclusive.

The coding region of each gene that comprises or is comprised by apolynucleotide sequence set forth in the Sequence Listing may beisolated by screening using a DNA sequence provided in the SequenceListing to synthesize an oligonucleotide probe. A labeledoligonucleotide having a sequence complementary to that of a gene of theinvention is then used to screen a library of cDNA, genomic DNA or mRNAto identify members of the library which hybridize to the probe. Forexample, synthetic oligonucleotides are prepared which correspond to theprenyltransferase or tocopherol cyclase EST sequences. Theoligonucleotides are used as primers in polymerase chain reaction (PCR)techniques to obtain 5′ and 3′ terminal sequence of prenyltransferase ortocopherol cyclase genes. Alternatively, where oligonucleotides of lowdegeneracy can be prepared from particular prenyltransferase ortocopherol cyclase peptides, such probes may be used directly to screengene libraries for prenyltransferase or tocopherol cyclase genesequences. In particular, screening of cDNA libraries in phage vectorsis useful in such methods due to lower levels of backgroundhybridization.

Typically, a prenyltransferase or tocopherol cyclase sequence obtainablefrom the use of nucleic acid probes will show 60-70% sequence identitybetween the target prenyltransferase or tocopherol cyclase sequence andthe encoding sequence used as a probe. However, lengthy sequences withas little as 50-60% sequence identity may also be obtained. The nucleicacid probes may be a lengthy fragment of the nucleic acid sequence, ormay also be a shorter, oligonucleotide probe. When longer nucleic acidfragments are employed as probes (greater than about 100 bp), one mayscreen at lower stringencies in order to obtain sequences from thetarget sample which have 20-50% deviation (i.e., 50-80% sequencehomology) from the sequences used as probe. Oligonucleotide probes canbe considerably shorter than the entire nucleic acid sequence encodingan prenyltransferase or tocopherol cyclase enzyme, but should be atleast about 10, preferably at least about 15, and more preferably atleast about 20 nucleotides. A higher degree of sequence identity isdesired when shorter regions are used as opposed to longer regions. Itmay thus be desirable to identify regions of highly conserved amino acidsequence to design oligonucleotide probes for detecting and recoveringother related prenyltransferase or tocopherol cyclase genes. Shorterprobes are often particularly useful for polymerase chain reactions(PCR), especially when highly conserved sequences can be identified.(See, Gould, et al., PNAS USA (1989) 86:1934-1938.).

Another aspect of the present invention relates to'prenyltransferase ortocopherol cyclase polypeptides. Such polypeptides include isolatedpolypeptides set forth in the Sequence Listing, as well as polypeptidesand fragments thereof, particularly those polypeptides which exhibitprenyltransferase or tocopherol cyclase activity and also thosepolypeptides which have at least 50%, 60% or 70% identity, preferably atleast 80% identity, more preferably at least 90% identity, and mostpreferably at least 95% identity to a polypeptide sequence selected fromthe group of sequences set forth in the Sequence Listing, and alsoinclude portions of such polypeptides, wherein such portion of thepolypeptide preferably includes at least 30 amino acids and morepreferably includes at least 50 amino acids.

“Identity”, as is well understood in the art, is a relationship betweentwo or more polypeptide sequences or two or more polynucleotidesequences, as determined by comparing the sequences. In the art,“identity” also means the degree of sequence relatedness betweenpolypeptide or polynucleotide sequences, as determined by the matchbetween strings of such sequences. “Identity” can be readily calculatedby known methods including, but not limited to, those described inComputational Molecular Biology, Lesk, A. M., ed., Oxford UniversityPress, New York (1988); Biocomputing: Informatics and Genome Projects,Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis ofSequence Data, Part I, Griffin, A. M. and Griffin, H. G., eds., HumanaPress, New Jersey (1994); Sequence Analysis in Molecular Biology, vonHeinje, G., Academic Press (1987); Sequence Analysis Primer, Gribskov,M. and Devereux, J., eds., Stockton Press, New York (1991); and Carillo,H., and Lipman, D., SIAM J Applied Math, 48:1073 (1988). Methods todetermine identity are designed to give the largest match between thesequences tested. Moreover, methods to determine identity are codifiedin publicly available programs. Computer programs which can be used todetermine identity between two sequences include, but are not limitedto, GCG (Devereux, J., et al., Nucleic Acids Research 12(1):387 (1984);suite of five BLAST programs, three designed for nucleotide sequencesqueries (BLASTN, BLASTX, and TBLASTX) and two designed for proteinsequence queries (BLASTP and TBLASTN) (Coulson, Trends in Biotechnology,12: 76-80 (1994); Birren, et al., Genome Analysis, 1: 543-559 (1997)).The BLAST X program is publicly available from NCBI and other sources(BLAST Manual, Altschul, S., et al., NCBI NLM NIH, Bethesda, Md. 20894;Altschul, S., et al., J. Mol. Biol., 215:403-410 (1990)). The well knownSmith Waterman algorithm can also be used to determine identity.

Parameters for polypeptide sequence comparison typically include thefollowing:

Algorithm: Needleman and Wunsch, J. Mol. Biol. 48:443-453 (1970)

Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc. Natl.Acad. Sci USA 89:10915-10919 (1992)

Gap Penalty: 12

Gap Length Penalty: 4

A program which can be used with these parameters is publicly availableas the “gap” program from Genetics Computer Group, Madison Wis. Theabove parameters along with no penalty for end gap are the defaultparameters for peptide comparisons.

Parameters for polynucleotide sequence comparison include the following:

Algorithm: Needleman and Wunsch, J. Mol. Biol. 48:443-453 (1970)

Comparison matrix: matches=+10; mismatches=0

Gap Penalty: 50

Gap Length Penalty: 3

A program which can be used with these parameters is publicly availableas the “gap” program from Genetics Computer Group, Madison Wis. Theabove parameters are the default parameters for nucleic acidcomparisons.

The invention also includes polypeptides of the formula:

X—(R₁)_(n)—(R₂)—(R₃)_(n)—Y

wherein, at the amino terminus, X is hydrogen, and at the carboxylterminus, Y is hydrogen or a metal, R₁ and R₃ are any amino acidresidue, n is an integer between 1 and 1000, and R₂ is an amino acidsequence of the invention, particularly an amino acid sequence selectedfrom the group set forth in the Sequence Listing and preferably thoseencoded by the sequences provided in SEQ ID NOs: 2, 4, 6, 9, 12, 17,19-22, 24-28, 30, 32-35, 37, and 39. In the formula, R₂ is oriented sothat its amino terminal residue is at the left, bound to R₁, and itscarboxy terminal residue is at the right, bound to R₃. Any stretch ofamino acid residues denoted by either R group, where R is greater than1, may be either a heteropolymer or a homopolymer, preferably aheteropolymer.

Polypeptides of the present invention include isolated polypeptidesencoded by a polynucleotide comprising a sequence selected from thegroup of a sequence contained in the Sequence Listing set forth herein.

The polypeptides of the present invention can be mature protein or canbe part of a fusion protein.

Fragments and variants of the polypeptides are also considered to be apart of the invention. A fragment is a variant polypeptide which has anamino acid sequence that is entirely the same as part but not all of theamino acid sequence of the previously described polypeptides. Thefragments can be “free-standing” or comprised within a largerpolypeptide of which the fragment forms a part or a region, mostpreferably as a single continuous region. Preferred fragments arebiologically active fragments which are those fragments that mediateactivities of the polypeptides of the invention, including those withsimilar activity or improved activity or with a decreased activity. Alsoincluded are those fragments that antigenic or immunogenic in an animal,particularly a human.

Variants of the polypeptide also include polypeptides that vary from thesequences set forth in the Sequence Listing by conservative amino acidsubstitutions, substitution of a residue by another with likecharacteristics. In general, such substitutions are among Ala, Val, Leuand Ile; between Ser and Thr; between Asp and Glu; between Asn and Gln;between Lys and Arg; or between Phe and Tyr. Particularly preferred arevariants in which 5 to 10; 1 to 5; 1 to 3 or one amino acid(s) aresubstituted, deleted, or added, in any combination.

Variants that are fragments of the polypeptides of the invention can beused to produce the corresponding full length polypeptide by peptidesynthesis. Therefore, these variants can be used as intermediates forproducing the full-length polypeptides of the invention.

The polynucleotides and polypeptides of the invention can be used, forexample, in the transformation of host cells, such as plant host cells,as further discussed herein.

The invention also provides polynucleotides that encode a polypeptidethat is a mature protein plus additional amino or carboxyl-terminalamino acids, or amino acids within the mature polypeptide (for example,when the mature form of the protein has more than one polypeptidechain). Such sequences can, for example, play a role in the processingof a protein from a precursor to a mature form, allow protein transport,shorten or lengthen protein half-life, or facilitate manipulation of theprotein in assays or production. It is contemplated that cellularenzymes can be used to remove any additional amino acids from the matureprotein.

A precursor protein, having the mature form of the polypeptide fused toone or more prosequences may be an inactive form of the polypeptide. Theinactive precursors generally are activated when the prosequences areremoved. Some or all of the prosequences may be removed prior toactivation. Such precursor protein are generally called proproteins.

Plant Constructs and Methods of Use

Of particular interest is the use of the nucleotide sequences inrecombinant DNA constructs to direct the transcription or transcriptionand translation (expression) of the prenyltransferase or tocopherolcyclase sequences of the present invention in a host plant cell. Theexpression constructs generally comprise a promoter functional in a hostplant cell operably linked to a nucleic acid sequence encoding aprenyltransferase or tocopherol cyclase of the present invention and atranscriptional termination region functional in a host plant cell.

A first nucleic acid sequence is “operably linked” or “operablyassociated” with a second nucleic acid sequence when the sequences areso arranged that the first nucleic acid sequence affects the function ofthe second nucleic-acid sequence. Preferably, the two sequences are partof a single contiguous nucleic acid molecule and more preferably areadjacent. For example, a promoter is operably linked to a gene if thepromoter regulates or mediates transcription of the gene in a cell.

Those skilled in the art will recognize that there are a number ofpromoters which are functional in plant cells, and have been describedin the literature. Chloroplast and plastid specific promoters,chloroplast or plastid functional promoters, and chloroplast or plastidoperable promoters are also envisioned.

One set of plant functional promoters are constitutive promoters such asthe CaMV35S or FMV35S promoters that yield high levels of expression inmost plant organs. Enhanced or duplicated versions of the CaMV35S andFMV35S promoters are useful in the practice of this invention (Odell, etal. (1985) Nature 313:810-812; Rogers, U.S. Pat. No. 5,378,619). Inaddition, it may also be preferred to bring about expression of theprenyltransferase or tocopherol cyclase gene in specific tissues of theplant, such as leaf, stem, root, tuber, seed, fruit, etc., and thepromoter chosen should have the desired tissue and developmentalspecificity.

Of particular interest is the expression of the nucleic acid sequencesof the present invention from transcription initiation regions which arepreferentially expressed in a plant seed tissue. Examples of such seedpreferential transcription initiation sequences include those sequencesderived from sequences encoding plant storage protein genes or fromgenes involved in fatty acid biosynthesis in oilseeds. Examples of suchpromoters include the 5′ regulatory regions from such genes as napin(Kridl et al., Seed Sci. Res. 1:209:219 (1991)), phaseolin, zein,soybean trypsin inhibitor, ACP, stearoyl-ACP desaturase, soybean α′subunit of β-conglycinin (soy 7s, (Chen et al.; Proc. Natl. Acad. Sci.,83:8560-8564 (1986))) and oleosin.

It may be advantageous to direct the localization of proteins conferringprenyltransferase or tocopherol cyclase to a particular subcellularcompartment, for example, to the mitochondrion, endoplasmic reticulum,vacuoles, chloroplast or other plastidic compartment. For example, wherethe genes of interest of the present invention will be targeted toplastids, such as chloroplasts, for expression, the constructs will alsoemploy the use of sequences to direct the gene to the plastid. Suchsequences are referred to herein as chloroplast transit peptides (CTP)or plastid transit peptides (PTP). In this manner, where the gene ofinterest is not directly inserted into the plastid, the expressionconstruct will additionally contain a gene encoding a transit peptide todirect the gene of interest to the plastid. The chloroplast transitpeptides may be derived from the gene of interest, or may be derivedfrom a heterologous sequence having a CTP. Such transit peptides areknown in the art. See, for example, Von Heijne et al. (1991) Plant Mol.Biol. Rep. 9:104-126; Clark et al. (1989) J. Biol. Chem.264:17544-17550; della-Cioppa et al. (1987) Plant Physiol. 84:965-968;Romer et al. (1993) Biochem. Biophys. Res Commun. 196:1414-1421; and,Shah et al. (1986) Science 233:478-481.

Depending upon the intended use, the constructs may contain the nucleicacid sequence which encodes the entire prenyltransferase or tocopherolcyclase protein, or a portion thereof. For example, where antisenseinhibition of a given prenyltransferase or tocopherol cyclase protein isdesired, the entire prenyltransferase or tocopherol cyclase sequence isnot required. Furthermore, where prenyltransferase or tocopherol cyclasesequences used in constructs are intended for use as probes, it may beadvantageous to prepare constructs containing only a particular portionof a prenyltransferase or tocopherol cyclase encoding sequence, forexample a sequence which is discovered to encode a highly conservedprenyltransferase or tocopherol cyclase region.

The skilled artisan will recognize that there are various methods forthe inhibition of expression of endogenous sequences in a host cell.Such methods include, but are not limited to, antisense suppression(Smith, et al. (1988) Nature 334:724-726), co-suppression (Napoli, etal. (1989) Plant Cell 2:279-289), ribozymes (PCT Publication WO97/10328), and combinations of sense and antisense Waterhouse, et al.(1998) Proc. Natl. Acad. Sci. USA 95:13959-13964. Methods for thesuppression of endogenous sequences in a host cell typically employ thetranscription or transcription and translation of at least a portion ofthe sequence to be suppressed. Such sequences may be homologous tocoding as well as non-coding regions of the endogenous sequence.

Regulatory transcript termination regions may be provided in plantexpression constructs of this invention as well. Transcript terminationregions may be provided by the DNA sequence encoding theprenyltransferase or tocopherol cyclase or a convenient transcriptiontermination region derived from a different gene source, for example,the transcript termination region which is naturally associated with thetranscript initiation region. The skilled artisan will recognize thatany convenient transcript termination region which is capable ofterminating transcription in a plant cell may be employed in theconstructs of the present invention.

Alternatively, constructs may be prepared to direct the expression ofthe prenyltransferase or tocopherol cyclase sequences directly from thehost plant cell plastid. Such constructs and methods are known in theart and are generally described, for example, in Svab, et al. (1990)Proc. Natl. Acad. Sci. USA 87:8526-8530 and Svab and Maliga (1993) Proc.Natl. Acad Sci. USA 90:913-917 and in U.S. Pat. No. 5,693,507.

The prenyltransferase or tocopherol cyclase constructs of the presentinvention can be used in transformation methods with additionalconstructs providing for the expression of other nucleic acid sequencesencoding proteins involved in the production of tocopherols, ortocopherol precursors such as homogentisic acid and/orphytylpyrophosphate. Nucleic acid sequences encoding proteins involvedin the production of homogentisic acid are known in the art, and includebut not are limited to, 4-hydroxyphenylpyruvate dioxygenase (HPPD, EC1.13.11.27) described for example, by Garcia, et al. ((1999) PlantPhysiol. 119(4):1507-1516), mono or bifunctional tyrA (described forexample by Xia, et al. (1992) J. Gen Microbiol. 138:1309-1316, andHudson, et al. (1984) J. Mol. Biol. 180:1023-1051), Oxygenase,4-hydroxyphenylpyruvate di-(9CI), 4-Hydroxyphenylpyruvate dioxygenase;p-Hydroxyphenylpyruvate dioxygenase; p-Hydroxyphenylpyruvatehydroxylase; p-Hydroxyphenylpyruvate oxidase; p-Hydroxyphenylpyruvicacid hydroxylase; p-Hydroxyphenylpyruvic hydroxylase;p-Hydroxyphenylpyruvic oxidase), 4-hydroxyphenylacetate, NAD(P)H:oxygenoxidoreductase (1-hydroxylating); 4-hydroxyphenylacetate1-monooxygenase, and the like. In addition, constructs for theexpression of nucleic acid sequences encoding proteins involved in theproduction of phytylpyrophosphate can also be employed with theprenyltransferase or tocopherol cyclase constructs of the presentinvention. Nucleic acid uences encoding proteins involved in theproduction of phytylpyrophosphate are known in the art, and include, butare not limited to geranylgeranylpyrophosphate synthase (GGPPS),geranylgeranylpyrophosphate reductase (GGH), 1-deoxyxylulose-5-phosphatesynthase, 1-deoxy-D-xylolose-5-phosphate reductoisomerase,4-diphosphocytidyl-2-C-methylerythritol synthase, isopentylpyrophosphate isomerase.

The prenyltransferase or tocopherol cyclase sequences of the presentinvention find use in the preparation of transformation constructshaving a second expression cassette for the expression of additionalsequences involved in tocopherol biosynthesis. Additional tocopherolbiosynthesis sequences of interest in the present invention include, butare not limited to gamma-tocpherol methyltransferase (Shintani, et al.(1998) Science 282(5396):2098-2100), tocopherol cyclase, and tocopherolmethyltransferase.

A plant cell, tissue, organ, or plant into which the recombinant DNAconstructs containing the expression constructs have been introduced isconsidered transformed, transfected, or transgenic. A transgenic ortransformed cell or plant also includes progeny of the cell or plant andprogeny produced from a breeding program employing such a transgenicplant as a parent in a cross and exhibiting an altered phenotyperesulting from the presence of a prenyltransferase or tocopherol cyclasenucleic acid sequence.

Plant expression or transcription constructs having a prenyltransferaseor tocopherol cyclase as the DNA sequence of interest for increased ordecreased expression thereof may be employed with a wide variety ofplant life, particularly, plant life involved in the production ofvegetable oils for edible and industrial uses. Particularly preferredplants for use in the methods of the present invention include, but arenot limited to: Acacia, alfalfa, aneth, apple, apricot, artichoke,arugula, asparagus, avocado, banana, barley, beans, beet, blackberry,blueberry, broccoli, brussels sprouts, cabbage, canola, cantaloupe,carrot, cassaya, cauliflower, celery, cherry, chicory, cilantro, citrus,clementines, coffee, corn, cotton, cucumber, Douglas fir, eggplant,endive, escarole, eucalyptus, fennel, figs, garlic, gourd, grape,grapefruit, honey dew, jicama, kiwifruit, lettuce, leeks, lemon, lime,Loblolly pine, mango, melon, mushroom, nectarine, nut, oat, oil palm,oil seed rape, okra, onion, orange, an ornamental plant, papaya,parsley, pea, peach, peanut, pear, pepper, persimmon, pine, pineapple,plantain, plum, pomegranate, poplar, potato, pumpkin, quince, radiatapine, radicchio, radish, raspberry, rice, rye, sorghum, Southern pine,soybean, spinach, squash, strawberry, sugarbeet, sugarcane, sunflower,sweet potato, sweetgum, tangerine, tea, tobacco, tomato, triticale,turf, turnip, a vine, watermelon, wheat, yams, and zucchini.

Most especially preferred are temperate oilseed crops. Temperate oilseedcrops of interest include, but are not limited to, rapeseed (Canola andHigh Erucic Acid varieties), sunflower, safflower, cotton, soybean,peanut, coconut and oil palms, and corn. Depending on the method forintroducing the recombinant constructs into the host cell, other DNAsequences may be required. Importantly, this invention is applicable todicotyledyons and monocotyledons species alike and will be readilyapplicable to new and/or improved transformation and regulationtechniques.

Of particular interest, is the use of prenyltransferase or tocopherolcyclase constructs in plants to produce plants or plant parts,including, but not limited to leaves, stems, roots, reproductive, andseed, with a modified content of tocopherols in plant parts havingtransformed plant cells.

For immunological screening, antibodies to the protein can be preparedby injecting rabbits or mice with the purified protein or portionthereof, such methods of preparing antibodies being well known to thosein the art. Either monoclonal or polyclonal antibodies can be produced,although typically polyclonal antibodies are more useful for geneisolation. Western analysis may be conducted to determine that a relatedprotein is present in a crude extract of the desired plant species, asdetermined by cross-reaction with the antibodies to the encodedproteins. When cross-reactivity is observed, genes encoding the relatedproteins are isolated by screening expression libraries representing thedesired plant species. Expression libraries can be constructed in avariety of commercially available vectors, including lambda gt11, asdescribed in Sambrook, et al. (Molecular Cloning: A Laboratory Manual,Second Edition (1989) Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y.).

To confirm the activity and specificity of the proteins encoded by theidentified nucleic acid sequences as prenyltransferase or tocopherolcyclase enzymes, in vitro assays are performed in insect cell culturesusing baculovirus expression systems. Such baculovirus expressionsystems are known in the art and are described by Lee, et al. U.S. Pat.No. 5,348,886, the entirety of which is herein incorporated byreference.

In addition, other expression constructs may be prepared to assay forprotein activity utilizing different expression systems. Such expressionconstructs are transformed into yeast or prokaryotic host and assayedfor prenyltransferase or tocopherol cyclase activity. Such expressionsystems are known in the art and are readily available throughcommercial sources.

In addition to the sequences described in the present invention, DNAcoding sequences useful in the present invention can be derived fromalgae, fungi, bacteria, mammalian sources, plants, etc. Homologysearches in existing databases using signature sequences correspondingto conserved nucleotide and amino acid sequences of prenyltransferase ortocopherol cyclase can be employed to isolate equivalent, related genesfrom other sources such as plants and microorganisms. Searches in ESTdatabases can also be employed. Furthermore, the use of DNA sequencesencoding enzymes functionally enzymatically equivalent to thosedisclosed herein, wherein such DNA sequences are degenerate equivalentsof the nucleic acid sequences disclosed herein in accordance with thedegeneracy of the genetic code, is also encompassed by the presentinvention. Demonstration of the functionality of coding sequencesidentified by any of these methods can be carried out by complementationof mutants of appropriate organisms, such as Synechocystis, Shewanella,yeast, Pseudomonas, Rhodobacteria, etc., that lack specific biochemicalreactions, or that have been mutated. The sequences of the DNA codingregions can be optimized by gene resynthesis, based on codon usage, formaximum expression in particular hosts.

For the alteration of tocopherol production in a host cell, a secondexpression construct can be used in accordance with the presentinvention. For example, the prenyltransferase or tocopherol cyclaseexpression construct can be introduced into a host cell in conjunctionwith a second expression construct having a nucleotide sequence for aprotein involved in tocopherol biosynthesis.

The method of transformation in obtaining such transgenic plants is notcritical to the instant invention, and various methods of planttransformation are currently available. Furthermore, as newer methodsbecome available to transform crops, they may also be directly appliedhereunder. For example, many plant species naturally susceptible toAgrobacterium infection may be successfully transformed via tripartiteor binary vector methods of Agrobacterium mediated transformation. Inmany instances, it will be desirable to have the construct bordered onone or both sides by T-DNA, particularly having the left and rightborders, more particularly the right border. This is particularly usefulwhen the construct uses A. tumefaciens or A. rhizogenes as a mode fortransformation, although the T-DNA borders may find use with other modesof transformation. In addition, techniques of microinjection, DNAparticle bombardment, and electroporation have been developed whichallow for the transformation of various monocot and dicot plant species.

Normally, included with the DNA construct will be a structural genehaving the necessary regulatory regions for expression in a host andproviding for selection of transformant cells. The gene may provide forresistance to a cytotoxic agent, e.g. antibiotic, heavy metal, toxin,etc., complementation providing prototrophy to an auxotrophic host,viral immunity or the like. Depending upon the number of different hostspecies the expression construct or components thereof are introduced,one or more markers may be employed, where different conditions forselection are used for the different hosts.

Where Agrobacterium is used for plant cell transformation, a vector maybe used which may be introduced into the Agrobacterium host forhomologous recombination with T-DNA or the Ti- or Ri-plasmid present inthe Agrobacterium host. The Ti- or Ri-plasmid containing the T-DNA forrecombination may be armed (capable of causing gall formation) ordisarmed (incapable of causing gall formation), the latter beingpermissible, so long as the vir genes are present in the transformedAgrobacterium host. The armed plasmid can give a mixture of normal plantcells and gall.

In some instances where Agrobacterium is used as the vehicle fortransforming host plant cells, the expression or transcription constructbordered by the T-DNA border region(s) will be inserted into a broadhost range vector capable of replication in E. coli and Agrobacterium,there being broad host range vectors described in the literature.Commonly used is pRK2 or derivatives thereof. See, for example, Ditta,et al., (Proc. Nat. Acad. Sci., U.S.A. (1980) 77:7347-7351) and EPA 0120 515, which are incorporated herein by reference. Alternatively, onemay insert the sequences to be expressed in plant cells into a vectorcontaining separate replication sequences, one of which stabilizes thevector in E. coli, and the other in Agrobacterium. See, for example,McBride, et al. (Plant Mol. Biol. (1990)/4:269-276), wherein the pRiHRI(Jouanin, et al., Mol. Gen. Genet. (1985) 201:370-374) origin ofreplication is utilized and provides for added stability of the plantexpression vectors in host Agrobacterium cells.

Included with the expression construct and the T-DNA will be one or moremarkers, which allow for selection of transformed Agrobacterium andtransformed plant cells. A number of markers have been developed for usewith plant cells, such as resistance to chloramphenicol, kanamycin, theaminoglycoside G418, hygromycin, or the like. The particular markeremployed is not essential to this invention, one or another marker beingpreferred depending on the particular host and the manner ofconstruction.

For transformation of plant cells using Agrobacterium, explants may becombined and incubated with the transformed Agrobacterium for sufficienttime for transformation, the bacteria killed, and the plant cellscultured in an appropriate selective medium. Once callus forms, shootformation can be encouraged by employing the appropriate plant hormonesin accordance with known methods and the shoots transferred to rootingmedium for regeneration of plants. The plants may then be grown to seedand the seed used to establish repetitive generations and for isolationof vegetable oils.

There are several possible ways to obtain the plant cells of thisinvention which contain multiple expression constructs. Any means forproducing a plant comprising a construct having a DNA sequence encodingthe expression construct of the present invention, and at least oneother construct having another DNA sequence encoding an enzyme areencompassed by the present invention. For example, the expressionconstruct can be used to transform a plant at the same time as thesecond construct either by inclusion of both expression constructs in asingle transformation vector or by using separate vectors, each of whichexpress desired genes. The second construct can be introduced into aplant which has already been transformed with the prenyltransferase ortocopherol cyclase expression construct, or alternatively, transformedplants, one expressing the prenyltransferase or tocopherol cyclaseconstruct and one expressing the second construct, can be crossed tobring the constructs together in the same plant.

Transgenic plants of the present invention may be produced from tissueculture, and subsequent generations grown from seed. Alternatively,transgenic plants may be grown using apomixis. Apomixis is a geneticallycontrolled method of reproduction in plants where the embryo is formedwithout union of an egg and a sperm. There are three basic types ofapomictic reproduction: 1) apospory where the embryo develops from achromosomally unreduced egg in an embryo sac derived from the nucleus,2) diplospory where the embryo develops from an unreduced egg in anembryo sac derived from the megaspore mother cell, and 3) adventitiousembryony where the embryo develops directly from a somatic cell. In mostforms of apomixis, pseudogamy or fertilization of the polar nuclei toproduce endosperm is necessary for seed viability. In apospory, a nursecultivar can be used as a pollen source for endosperm formation inseeds. The nurse cultivar does not affect the genetics of the aposporousapomictic cultivar since the unreduced egg of the cultivar developsparthenogenetically, but makes possible endosperm production. Apomixisis economically important, especially in transgenic plants, because itcauses any genotype, no matter how heterozygous, to breed true. Thus,with apomictic reproduction, heterozygous transgenic plants can maintaintheir genetic fidelity throughout repeated life cycles. Methods for theproduction of apomictic plants are known in the art. See, U.S. Pat. No.5,811,636, which is herein incorporated by reference in its entirety.

The nucleic acid sequences of the present invention can be used inconstructs to provide for the expression of the sequence in a variety ofhost cells, both prokaryotic eukaryotic. Host cells of the presentinvention preferably include monocotyledenous and dicotyledenous plantcells.

In general, the skilled artisan is familiar with the standard resourcematerials which describe specific conditions and procedures for theconstruction, manipulation and isolation of macromolecules (e.g., DNAmolecules, plasmids, etc.), generation of recombinant organisms and thescreening and isolating of clones, (see for example, Sambrook et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press (1989);Maliga et al., Methods in Plant Molecular Biology, Cold Spring HarborPress (1995), the entirety of which is herein incorporated by reference;Birren et al., Genome Analysis: Analyzing DNA, 1, Cold Spring Harbor,N.Y., the entirety of which is herein incorporated by reference).

Methods for the expression of sequences in insect host cells are knownin the art. Baculovirus expression vectors are recombinant insectviruses in which the coding sequence for a chosen foreign gene has beeninserted behind a baculovirus promoter in place of the viral gene, e.g.,polyhedrin (Smith and Summers, U.S. Pat. No. 4,745,051, the entirety ofwhich is incorporated herein by reference). Baculovirus expressionvectors are known in the art, and are described for example in Doerfler,Curr. Top. Microbiol. Immunol. 131:51-68 (1968); Luckow and Summers,Bio/Technology 6:47-55 (1988a); Miller, Annual Review of Microbiol.42:177-199 (1988); Summers, Curr. Comm. Molecular Biology, Cold SpringHarbor Press, Cold Spring Harbor, N.Y. (1988); Summers and Smith, AManual of Methods for Baculovirus Vectors and Insect Cell CultureProcedures, Texas Ag. Exper. Station Bulletin No. 1555 (1988), theentireties of which is herein incorporated by reference)

Methods for the expression of a nucleic acid sequence of interest in afungal host cell are known in the art. The fungal host cell may, forexample, be a yeast cell or a filamentous fungal cell. Methods for theexpression of DNA sequences of interest in yeast cells are generallydescribed in “Guide to yeast genetics and molecular biology”, Guthrieand Fink, eds. Methods in enzymology, Academic Press, Inc. Vol 194(1991) and Gene expression technology”, Goeddel ed, Methods inEnzymology, Academic Press, Inc., Vol 185 (1991).

Mammalian cell lines available as hosts for expression are known in theart and include many immortalized cell lines available from the AmericanType Culture Collection (ATCC, Manassas, Va.), such as HeLa cells,Chinese hamster ovary (CHO) cells, baby hamster kidney (BHK) cells and anumber of other cell lines. Suitable promoters for mammalian cells arealso known in the art and include, but are not limited to, viralpromoters such as that from Simian Virus 40 (SV40) (Fiers et al., Nature273:113 (1978), the entirety of which is herein incorporated byreference), Rous sarcoma virus (RSV), adenovirus (ADV) and bovinepapilloma virus (BPV). Mammalian cells may also require terminatorsequences and poly-A addition sequences. Enhancer sequences whichincrease expression may also be included and sequences which promoteamplification of the gene may also be desirable (for examplemethotrexate resistance genes).

Vectors suitable for replication in mammalian cells are well known inthe art, and may include viral replicons, or sequences which insureintegration of the appropriate sequences encoding epitopes into the hostgenome. Plasmid vectors that greatly facilitate the construction ofrecombinant viruses have been described (see, for example, Mackett etal, J. Virol. 49:857 (1984); Chakrabarti et al., Mol. Cell. Biol. 5:3403(1985); Moss, In: Gene Transfer Vectors For Mammalian Cells (Miller andCalos, eds., Cold Spring Harbor Laboratory, N.Y., p. 10, (1987); all ofwhich are herein incorporated by reference in their entirety).

The invention also includes plants and plant parts, such as seed, oiland meal derived from seed, and feed and food products processed fromplants, which are enriched in tocopherols. Of particular interest isseed oil obtained from transgenic plants where the tocopherol level hasbeen increased as compared to seed oil of a non-transgenic plant.

The harvested plant material may be subjected to additional processingto further enrich the tocopherol content. The skilled artisan willrecognize that there are many such processes or methods for refining,bleaching and degumming oil. U.S. Pat. No. 5,932,261, issued Aug. 3,1999, discloses on such process, for the production of a naturalcarotene rich refined and deodorised oil by subjecting the oil to apressure of less than 0.060 mbar and to a temperature of less than200.degree. C. Oil distilled by this process has reduced free fattyacids, yielding a refined, deodorised oil where Vitamin E contained inthe feed oil is substantially retained in the processed oil. Theteachings of this patent are incorporated herein by reference.

The invention now being generally described, it will be more readilyunderstood by reference to the following examples which are included forpurposes of illustration only and are not intended to limit the presentinvention.

EXAMPLES Example 1 Identification of Prenyltransferase or TocopherolCyclase Sequences

PSI-BLAST (Altschul, et al. (1997) Nuc Acid Res 25:3389-3402) profileswere generated for both the straight chain and aromatic classes ofprenyltransferases. To generate the straight chain profile, aprenyl-transferase from Porphyra purpurea (Genbank accession 1709766)was used as a query against the NCBI non-redundant protein database. TheE. coli enzyme involved in the formation of ubiquinone, ubiA (genbankaccession 1790473) was used as a starting sequence to generate thearomatic prenyltransferase profile. These profiles were used to searchpublic and proprietary DNA and protein data bases. In Arabidopsis sixputative prenyltransferases of the straight-chain class were identified,ATPT1, (SEQ ID NO:9), ATPT7 (SEQ ID NO:10), ATPT8 (SEQ ID NO:11), ATPT9(SEQ ID NO:13), ATPT10 (SEQ ID NO:14), and ATPT11 (SEQ ID NO:15), andsix were identified of the aromatic class, ATPT2 (SEQ ID NO:1), ATPT3(SEQ ID NO:3), ATPT4 (SEQ ID NO:5), ATPT5 (SEQ ID NO:7), ATPT6 (SEQ IDNO:8), and ATPT12 (SEQ ID NO:16). Additional prenyltransferase sequencesfrom other plants related to the aromatic class of prenyltransferases,such as soy (SEQ ID NOs: 19-23, the deduced amino acid sequence of SEQID NO:23 is provided in SEQ ID NO:24) and maize (SEQ ID NOs: 25-29, and31) are also identified. The deduced amino acid sequence of ZMPT5 (SEQID NO:29) is provided in SEQ ID NO:30.

Searches are performed on a Silicon Graphics Unix computer usingadditional Bioaccellerator hardware and GenWeb software supplied byCompugen Ltd. This software and hardware enables the use of theSmith-Waterman algorithm in searching DNA and protein databases usingprofiles as queries. The program used to query protein databases isprofilesearch. This is a search where the query is not a single sequencebut a profile based on a multiple alignment of amino acid or nucleicacid sequences. The profile is used to query a sequence data set, i.e.,a sequence database. The profile contains all the pertinent informationfor scoring each position in a sequence, in effect replacing the“scoring matrix” used for the standard query searches. The program usedto query nucleotide databases with a protein profile is tprofilesearch.Tprofilesearch searches nucleic acid databases using an amino acidprofile query. As the search is running, sequences in the database aretranslated to amino acid sequences in six reading frames. The outputfile for tprofilesearch is identical to the output file forprofilesearch except for an additional column that indicates the framein which the best alignment occurred.

The Smith-Waterman algorithm, (Smith and Waterman (1981) supra), is usedto search for similarities between one sequence from the query and agroup of sequences contained in the database. E score values as well asother sequence information, such as conserved peptide sequences are usedto identify related sequences.

To obtain the entire coding region corresponding to the Arabidopsisprenyltransferase sequences, synthetic oligo-nucleotide primers aredesigned to amplify the 5′ and 3′ ends of partial cDNA clones containingprenyltransferase sequences. Primers are designed according to therespective Arabidopsis prenyltransferase sequences and used in RapidAmplification of cDNA Ends (RACE) reactions (Frohman et al. (1988) Proc.Natl. Acad. Sci. USA 85:8998-9002) using the Marathon cDNA amplificationkit (Clontech Laboratories Inc, Palo Alto, Calif.).

Amino acid sequence alignments between ATPT2 (SEQ ID NO:2), ATPT3 (SEQID NO:4), ATPT4 (SEQ ID NO:6), ATPT8 (SEQ ID NO:12), and ATPT12 (SEQ IDNO:17) are performed using ClustalW (FIG. 1), and the percent identityand similarities are provided in Table 1 below.

TABLE 1: ATPT2 ATPT3 ATPT4 ATPT8 ATPT12 ATPT2 % Identity 12 13 11 15 %similar 25 25 22 32 % Gap 17 20 20 9 ATPT3 % Identity 12 6 22 % similar29 16 38 % Gap 20 24 14 ATPT4 % Identity 9 14 % similar 18 29 % Gap 2619 ATPT8 % Identity 7 % similar 19 % Gap 20 ATPT12 % Identity % similar% Gap

Example 2 Preparation of Prenyl Transferase Expression Constructs

A plasmid containing the napin cassette derived from pCGN3223 (describedin U.S. Pat. No. 5,639,790, the entirety of which is incorporated hereinby reference) was modified to make it more useful for cloning large DNAfragments containing multiple restriction sites, and to allow thecloning of multiple napin fusion genes into plant binary transformationvectors. An adapter comprised of the self annealed oligonucleotide ofsequence CGCGATTTAAATGGCGCGCCCTGCAGGCGGCCGCCTGCAGGGCGCGCCATTTAAAT (SEQID NO:40) was ligated into the cloning vector pBC SK+ (Stratagene) afterdigestion with the restriction endonuclease BssHII to construct vectorpCGN7765. Plamids pCGN3223 and pCGN7765 were digested with NotI andligated together. The resultant vector, pCGN7770, contains the pCGN7765backbone with the napin seed specific expression cassette from pCGN3223.

The cloning cassette, pCGN7787, essentially the same regulatory elementsas pCGN7770, with the exception of the napin regulatory regions ofpCGN7770 have been replaced with the double CAMV 35S promoter and thetml polyadenylation and transcriptional termination region.

A binary vector for plant transformation, pCGN5139, was constructed frompCGN1558 (McBride and Summerfelt, (1990) Plant Molecular Biology,14:269-276). The polylinker of pCGN1558 was replaced as a HindIII/Asp718fragment with a polylinker containing unique restriction endonucleasesites, AscI, Pad, XbaI, SwaI, BamHI, and NotI. The Asp718 and HindIIIrestriction endonuclease sites are retained in pCGN5139.

A series of turbo binary vectors are constructed to allow for the rapidcloning of DNA sequences into binary vectors containing transcriptionalinitiation regions (promoters) and transcriptional termination regions.

The plasmid pCGN8618 was constructed by ligating oligonucleotides5′-TCGAGGATCCGCGGCCGCAAGCTTCCTGCAGG-3′ (SEQ ID NO:41) and5′-TCGACCTGCAGGAAGCTTGCGGCCGCGGATCC-3′ (SEQ ID NO:42) intoSalI/XhoI-digested pCGN7770. A fragment containing the napin promoter,polylinker and napin 3′ region was excised from pCGN8618 by digestionwith Asp718I; the fragment was blunt-ended by filling in the 5′overhangs with Klenow fragment then ligated into pCGN5139 that had beendigested with Asp718I and HindIII and blunt-ended by filling in the 5′overhangs with Klenow fragment. A plasmid containing the insert orientedso that the napin promoter was closest to the blunted Asp718I site ofpCGN5139 and the napin 3′ was closest to the blunted HindIII site wassubjected to sequence analysis to confirm both the insert orientationand the integrity of cloning junctions. The resulting plasmid wasdesignated pCGN8622.

The plasmid pCGN8619 was constructed by ligating oligonucleotides5′-TCGACCTGCAGGAAGCTTGCGGCCGCGGATCC-3′ (SEQ ID NO:43) and5′-TCGAGGATCCGCGGCCGCAAGCTTCCTGCAGG-3′ (SEQ ID NO:44) intoSalI/XhoI-digested pCGN7770. A fragment containing the napin promoter,polylinker and napin 3′ region was removed from pCGN8619 by digestionwith Asp718I; the fragment was blunt-ended by filling in the 5′overhangs with Klenow fragment then ligated into pCGN5139 that had beendigested with Asp718I and HindIII and blunt-ended by filling in the 5′overhangs with Klenow fragment. A plasmid containing the insert orientedso that the napin promoter was closest to the blunted Asp718I site ofpCGN5139 and the napin 3′ was closest to the blunted HindIII site wassubjected to sequence analysis to confirm both the insert orientationand the integrity of cloning junctions. The resulting plasmid wasdesignated pCGN8623.

The plasmid pCGN8620 was constructed by ligating oligonucleotides5′-TCGAGGATCCGCGGCCGCAAGCTTCCTGCAGGAGCT-3′ (SEQ ID NO:45) and5′-CCTGCAGGAAGCTTGCGGCCGCGGATCC-3′ (SEQ ID NO:46) intoSalI/SacI-digested pCGN7787. A fragment containing the d35S promoter,polylinker and tml 3′ region was removed from pCGN8620 by completedigestion with Asp718I and partial digestion with NotI. The fragment wasblunt-ended by filling in the 5′ overhangs with Klenow fragment thenligated into pCGN5139 that had been digested with Asp718I and HindIIIand blunt-ended by filling in the 5′ overhangs with Klenow fragment. Aplasmid containing the insert oriented so that the d35S promoter wasclosest to the blunted Asp718I site of pCGN5139 and the tml 3′ wasclosest to the blunted HindIII site was subjected to sequence analysisto confirm both the insert orientation and the integrity of cloningjunctions. The resulting plasmid was designated pCGN8624.

The plasmid pCGN8621 was constructed by ligating oligonucleotides5′-TCGACCTGCAGGAAGCTTGCGGCCGCGGATCCAGCT-3′ (SEQ ID NO:47) and5′-GGATCCGCGGCCGCAAGCTTCCTGCAGG-3′ (SEQ ID NO:48) intoSalI/SacI-digested pCGN7787. A fragment containing the d35S promoter,polylinker and tml 3′ region was removed from pCGN8621 by completedigestion with Asp718I and partial digestion with NotI. The fragment wasblunt-ended by filling in the 5′ overhangs with Klenow fragment thenligated into pCGN5139 that had been digested with Asp718I and HindIIIand blunt-ended by filling in the 5′ overhangs with Klenow fragment. Aplasmid containing the insert oriented so that the d35S promoter wasclosest to the blunted Asp718I site of pCGN5139 and the tml 3′ wasclosest to the blunted HindIII site was subjected to sequence analysisto confirm both the insert orientation and the integrity of cloningjunctions. The resulting plasmid was designated pCGN8625.

The plasmid construct pCGN8640 is a modification of pCGN8624 describedabove. A 938 bp PstI fragment isolated from transposon Tn7 which encodesbacterial spectinomycin and streptomycin resistance (Fling et al.(1985), Nucleic Acids Research 13(19):7095-7106), a determinant for E.coli and Agrobacterium selection, was blunt ended with Pfu polymerase.The blunt ended fragment was ligated into pCGN8624 that had beendigested with SpeI and blunt ended with Pfu polymerase. The regioncontaining the PstI fragment was sequenced to confirm both the insertorientation and the integrity of cloning junctions.

The spectinomycin resistance marker was introduced into pCGN8622 andpCGN8623 as follows. A 7.7 Kbp AvrII-SnaBI fragment from pCGN8640 wasligated to a 10.9 Kbp AvrII-SnaBI fragment from pCGN8623 or pCGN8622,described above. The resulting plasmids were pCGN8641 and pCGN8643,respectively.

The plasmid pCGN8644 was constructed by ligating oligonucleotides5′-GATCACCTGCAGGAAGCTTGCGGCCGCGGATCCAATGCA-3′ (SEQ ID NO:49) and5′-TTGGATCCGCGGCCGCAAGCTTCCTGCAGGT-3′ (SEQ ID NO:50) into BamHI-PstIdigested pCGN8640.

Synthetic oligonulceotides were designed for use in Polymerase ChainReactions (PCR) to amplify the coding sequences of ATPT2, ATPT3, ATPT4,ATPT8, and ATPT12 for the preparation of expression constructs and areprovided in Table 2 below.

TABLE 2 SEQ Restriction ID Name Site Sequence NO: ATPT2 5′ NotIGGATCCGCGGCCGCACAATGGAGTC 51 TCTGCTCTCTAGTTCT ATPT2 3′ SseIGGATCCTGCAGGTCACTTCAAAAAA 52 GGTAACAGCAAGT ATPT3 5′ NotIGGATCCGCGGCCGCACAATGGCGTT 53 TTTTGGGCTCTCCCGTGTTT ATPT3 3′ SseIGGATCCTGCAGGTTATTGAAAACTT 54 CTTCCAAGTACAACT ATPT4 5′ NotIGGATCCGCGGCCGCACAATGTGGCG 55 AAGATCTGTTGTT ATPT4 3′ SseIGGATCCTGCAGGTCATGGAGAGTAG 56 AAGGAAGGAGCT ATPT8 5′ NotIGGATCCGCGGCCGCACAATGGTACT 57 TGCCGAGGTTCCAAAGCTTGCCTCT ATPT8 3′ SseIGGATCCTGCAGGTCACTTGTTTCTG 58 GTGATGACTCTAT ATPT12 5′ NotIGGATCCGCGGCCGCACAATGACTTC 59 GATTCTCAACACT ATPT12 3′ SseIGGATCCTGCAGGTCAGTGTTGCGAT 60 GCTAATGCCGT

The coding sequences of ATPT2, ATPT3, ATPT4, ATPT8, and ATPT12 were allamplified using the respective PCR primers shown in Table 2 above andcloned into the TopoTA vector (Invitrogen). Constructs containing therespective prenyltransferase sequences were digested with NotI andSse8387I and cloned into the turbobinary vectors described above.

The sequence encoding ATPT2 prenyltransferase was cloned in the senseorientation into pCGN8640 to produce the plant transformation constructpCGN10800 (FIG. 2). The ATPT2 sequence is under control of the 35Spromoter.

The ATPT2 sequence was also cloned in the antisense orientation into theconstruct pCGN8641 to create pCGN10801 (FIG. 3). This construct providesfor the antisense expression of the ATPT2 sequence from the napinpromoter.

The ATPT2 coding sequence was also cloned in the sense orientation intothe vector pCGN8643 to create the plant transformation constructpCGN10822

The ATPT2 coding sequence was also cloned in the antisense orientationinto the vector pCGN8644 to create the plant transformation constructpCGN10803 (FIG. 4).

The ATPT4 coding sequence was cloned into the vector pCGN864 to createthe plant transformation construct pCGN10806 (FIG. 5). The ATPT2 codingsequence was cloned into the vector TopoTA™ vector from Invitrogen, tocreate the plant transformation construct pCGN10807 (FIG. 6). The ATPT3coding sequence was cloned into the TopoTA vector to create the planttransformation construct pCGN10808 (FIG. 7). The ATPT3 coding sequencewas cloned in the sense orientation into the vector pCGN8640 to createthe plant transformation construct pCGN10809 (FIG. 8). The ATPT3 codingsequence was cloned in the antisense orientation into the vectorpCGN8641 to create the plant transformation construct pCGN10810 (FIG.9). The ATPT3 coding sequence was cloned into the vector pCGN8643 tocreate the plant transformation construct pCGN10811 (FIG. 10). The ATPT3coding sequence was cloned into the vector pCGN8644 to create the planttransformation construct pCGN10812 (FIG. 11). The ATPT4 coding sequencewas cloned into the vector pCGN8640 to create the plant transformationconstruct pCGN10813 (FIG. 12). The ATPT4 coding sequence was cloned intothe vector pCGN8641 to create the plant transformation constructpCGN10814 (FIG. 13). The ATPT4 coding sequence was cloned into thevector pCGN8643 to create the plant transformation construct pCGN10815(FIG. 14). The ATPT4 coding sequence was cloned in the antisenseorientation into the vector pCGN8644 to create the plant transformationconstruct pCGN10816 (FIG. 15). The ATPT8 coding sequence was cloned inthe sense orientation into the vector pCGN8643 to create the planttransformation construct pCGN10819 (FIG. 17). The ATPT12 coding sequencewas cloned into the vector pCGN8640 to create the plant transformationconstruct pCGN10824 (FIG. 18). The ATPT12 coding sequence was clonedinto the vector pCGN8643 to create the plant transformation constructpCGN10825 (FIG. 19). The ATPT8 coding sequence was cloned into thevector pCGN8640 to create the plant transformation construct pCGN10826(FIG. 20).

Example 3 Plant Transformation with Prenyl Transferase Constructs

Transgenic Brassica plants are obtained by Agrobacterium-mediatedtransformation as described by Radke et al. (Theor. Appl. Genet. (1988)75:685-694; Plant Cell Reports (1992) 11:499-505). TransgenicArabidopsis thaliana plants may be obtained by Agrobacterium-mediatedtransformation as described by Valverkens et al., (Proc. Nat. Acad. Sci.(1988) 85:5536-5540), or as described by Bent et al. ((1994), Science265:1856-1860), or Bechtold et al. ((1993), C. R. Acad. Sci, LifeSciences 316:1194-1199). Other plant species may be similarlytransformed using related techniques.

Alternatively, microprojectile bombardment methods, such as described byKlein et al. (Bio/Technology 10:286-291) may also be used to obtainnuclear transformed plants.

Example 4 Identification of Additional Prenyltransferases

Additional BLAST searches were performed using the ATPT2 sequence, asequence in the class of aromatic prenyltransferases. ESTs, and in somecase, full-length coding regions, were identified in proprietary DNAlibraries.

Soy full-length homologs to ATPT2 were identified by a combination ofBLAST (using ATPT2 protein sequence) and 5′ RACE. Two homologs resulted(SEQ ID NO:95 and SEQ ID NO:96). Translated amino acid sequences areprovided by SEQ ID NO:97 and SEQ ID NO:98.

A rice est ATPT2 homolog is shown in SEQ ID NO:99 (obtained from BLASTusing the wheat ATPT2 homolog).

Other homolog sequences were obtained using ATPT2 and PSI-BLAST,including est sequences from wheat (SEQ ID NO:100), leek (SEQ ID NOs:101and 102), canola (SEQ ID NO:103), corn (SEQ ID NOs:104, 105 and 106),cotton (SEQ ID NO:107) and tomato (SEQ ID NO:108).

A PSI-Blast profile generated using the E. coli ubiA (genbank accession1790473) sequence was used to analyze the Synechocystis genome. Thisanalysis identified 5 open reading frames (ORFs) in the Synechocystisgenome that were potentially prenyltransferases; slr0926 (annotated asubiA (4-hydroxybenzoate-octaprenyltransferase, SEQ ID NO:32), sll1899(annotated as ctaB (cytocrome c oxidase folding protein, SEQ ID NO:33),slr0056 (annotated as g4 (chlorophyll synthase 33 kd subunit, SEQ IDNO:34), slr1518 (annotated as menA (menaquinone biosynthesis protein,SEQ ID NO:35), and slr1736 (annotated as a hypothetical protein ofunknown function (SEQ ID NO:36).

4A. Synechocystis Knock-Outs

To determine the functionality of these ORFs and their involvement, ifany, in the biosynthesis of tocopherols, knockouts constructs were madeto disrupt the ORF identified in Synechocystis.

Synthetic oligos were designed to amplify regions from the 5′(5′-TAATGTGTACATTGTCGGCCTC (17365′) (SEQ ID NO:61) and 5%GCAATGTAACATCAGAGATTTTGAGACACAACGTGGCTTTCCACAATTCCCCGCACC GTC(1736kanpr1)) (SEQ ID NO:62) and 3′ (5′-AGGCTAATAAGCACAAATGGGA (17363′)(SEQ ID NO:63) and 5′-GGTATGAGTCAGCAACACCTTCTTCACGAGGCAGACCTCAGCGGAATTGGTTTAGGTTATCCC (1736kanpr2)) (SEQ ID NO:64) ends of the slr1736ORF.

The 1736kanpr1 and 1736kanpr2 oligos contained 20 by of homology to theslr1736 ORF with an additional 40 by of sequence homology to the ends ofthe kanamycin resistance cassette. Separate PCR steps were completedwith these oligos and the products were gel purified and combined withthe kanamycin resistance gene from puc4K (Pharmacia) that had beendigested with HincII and gel purified away from the vector backbone. Thecombined fragments were allowed to assemble without oligos under thefollowing conditions: 94° C. for 1 min, 55° C. for 1 min, 72° C. for 1min plus 5 seconds per cycle for 40 cycles using pfu polymerase in 100ulreaction volume (Zhao, H and Arnold (1997) Nucleic Acids Res.25(6):1307-1308). One microliter or five microliters of this assemblyreaction was then amplified using 5′ and 3′ oligos nested within theends of the ORF fragment, so that the resulting product contained100-200 by of the 5′ end of the Synechocystis gene to be knocked out,the kanamycin resistance cassette, and 100-200 by of the 3′ end of thegene to be knocked out. This PCR product was then cloned into the vectorpGemT easy (Promega) to create the construct pMON21681 and used forSynechocystis transformation.

Primers were also synthesized for the preparation of Synechocystisknockout constructs for the other sequences using the same method asdescribed above, with the following primers. The ubiA 5′ sequence wasamplified using the primers 5′-GGATCCATGGTT GCCCAAACCCCATC (SEQ IDNO:65) and 5′-GCAATGTAACATCAGAGA TTTTGAGACACAACGTGGCTTTGGGTAAGCAACAATGACCGGC (SEQ ID NO:66). The 3′ region was amplifiedusing the synthetic oligonucleotide primers 5′-GAATTCTCAAAGCCAGCCCAGTAAC(SEQ ID NO:67) and 5′-GGTATGAGTCAGCAACACCTTCTTCACGAGGCAGACCTCAGCGGGTGCGAAAAGGGTTTTCCC (SEQ ID NO:68).The amplification products were combined with the kanamycin resistancegene from puc4K (Pharmacia) that had been digested with Hindi and gelpurified away from the vector backbone. The annealed fragment wasamplified using 5′ and 3′ oligos nested within the ends of the ORFfragment (5′-CCAGTGGTTTAGGCTGTGTGGTC (SEQ ID NO:69) and5′-CTGAGTTGGATGTATTGGATC (SEQ ID NO:70)), so that the resulting productcontained 100-200 by of the 5′ end of the Synechocystis gene to beknocked out, the kanamycin resistance cassette, and 100-200 by of the 3′end of the gene to be knocked out. This PCR product was then cloned intothe vector pGemT easy (Promega) to create the construct pMON21682 andused for Synechocystis transformation.

Primers were also synthesized for the preparation of Synechocystisknockout constructs for the other sequences using the same method asdescribed above, with the following primers. The sl11899 5′ sequence wasamplified using the primers 5′-GGATCCATGGTTACTT CGACAAAAATCC (SEQ IDNO:71) and 5′-GCAATGTAACATCAGAGATTTTGAGACACAACGTGGCTTTGCTAGGCAACCGCTTAGTAC (SEQ ID NO:72). The 3′region was amplified using the synthetic oligonucleotide primers5′-GAATTCTTAACCCAACAGTAAAGTTCCC (SEQ ID NO:73) and 5′-GGTATGAGTCAGCAACACCTTCTTCACGAGGCAGACCTCAGCGCCGGCATTGTCTTTTACATG (SEQ ID NO:74). Theamplification products were combined with the kanamycin resistance genefrom puc4K (Pharmacia) that had been digested with HincII and gelpurified away from the vector backbone. The annealed fragment wasamplified using 5′ and 3′ oligos nested within the ends of the ORFfragment (5′-GGAACCCTTGCAGCCGCTTC (SEQ ID NO:75) and5′-GTATGCCCAACTGGTGCAGAGG (SEQ ID NO:76)), so that the resulting productcontained 100-200 by of the 5′ end of the Synechocystis gene to beknocked out, the kanamycin resistance cassette, and 100-200 by of the 3′end of the gene to be knocked out. This PCR product was then cloned intothe vector pGemT easy (Promega) to create the construct pMON21679 andused for Synechocystis transformation.

Primers were also synthesized for the preparation of Synechocystisknockout constructs for the other sequences using the same method asdescribed above, with the following primers. The slr0056 5′ sequence wasamplified using the primers 5′-GGATCCATGTCTGACACACAAAATACCG (SEQ IDNO:77) and 5′-GCAATGTAACATCAGAGATTTTGAGACACAACGTGGCTTTCGCCAATACCAGCCACCAACAG (SEQ ID NO:78). The 3′ region was amplified using the syntheticoligonucleotide primers 5′-GAATTCTCAAAT CCCCGCATGGCCTAG (SEQ ID NO:79)and 5% GGTATGAGTCAGCAACACCTTCTTCACGAGGCAGACCTCAGCGGCCTACGGCTTGGACGTGTGGG (SEQ ID NO:80). The amplification products were combined withthe kanamycin resistance gene from puc4K (Pharmacia) that had beendigested with HindIII and gel purified away from the vector backbone.The annealed fragment was amplified using 5′ and 3′ oligos nested withinthe ends of the ORF fragment (5′-CACTTGGATTCCCCTGATCTG (SEQ ID NO:81)and 5′-GCAATACCCGCTTGGAAAACG (SEQ ID NO:82)), so that the resultingproduct contained 100-200 by of the 5′ end of the Synechocystis gene tobe knocked out, the kanamycin resistance cassette, and 100-200 by of the3′ end of the gene to be knocked out. This PCR product was then clonedinto the vector pGemT easy (Promega) to create the construct pMON21677and used for Synechocystis transformation.

Primers were also synthesized for the preparation of Synechocystisknockout constructs for the other sequences using the same method asdescribed above, with the following primers. The slr1518 5′ sequence wasamplified using the primers 5′-GGATCCATGACCGAAT CTTCGCCCCTAGC (SEQ IDNO:83) and 5′-GCAATGTAACATCAGAGATTTTGA GACACAACGTGGCTTTCAATCCTAGGTAGCCGAGGCG (SEQ ID NO:84). The 3′ region was amplifiedusing the synthetic oligonucleotide primers 5′-GAATTCTTAGCCCAGGCCAGCCCAGCC (SEQ ID NO:85) and 5′-GGTATGAGTCAGCAACACCTTCTTCACGAGGCAGACCTCAGCGGGGAATTGATTTGTTTAATTACC (SEQ ID NO:86). The amplificationproducts were combined with the kanamycin resistance gene from puc4K(Pharmacia) that had been digested with HincII and gel purified awayfrom the vector backbone. The annealed fragment was amplified using 5′and 3′ oligos nested within the ends of the ORF fragment(5′-GCGATCGCCATTATCGCTTGG (SEQ ID NO:87) and 5′-GCAGACTGGCAATTATCAGTAACG(SEQ ID NO:88)), so that the resulting product contained 100-200 by ofthe 5′ end of the Synechocystis gene to be knocked out, the kanamycinresistance cassette, and 100-200 by of the 3′ end of the gene to beknocked out. This PCR product was then cloned into the vector pGemT easy(Promega) to create the construct pMON21680 and used for Synechocystistransformation.

4B. Transformation of Synechocystis

Cells of Synechocystis 6803 were grown to a density of approximately2×10⁸ cells per ml and harvested by centrifugation. The cell pellet wasre-suspended in fresh BG-11 medium (ATCC Medium 616) at a density of1×10⁹ cells per ml and used immediately for transformation. One-hundredmicroliters of these cells were mixed with 5 ul of mini prep DNA andincubated with light at 30 C for 4 hours. This mixture was then platedonto nylon filters resting on BG-11 agar supplemented with TES pH8 andallowed to grow for 12-18 hours. The filters were then transferred toBG-11 agar+TES+5 ug/ml kanamycin and allowed to grow until coloniesappeared within 7-10 days (Packer and Glazer, 1988). Colonies were thenpicked into BG-11 liquid media containing 5 ug/ml kanamycin and allowedto grow for 5 days. These cells were then transferred to Bg-11 mediacontaining 10 ug/ml kanamycin and allowed to grow for 5 days and thentransferred to Bg-11+kanamycin at 25 ug/ml and allowed to grow for 5days. Cells were then harvested for PCR analysis to determine thepresence of a disrupted ORF and also for HPLC analysis to determine ifthe disruption had any effect on tocopherol levels.

PCR analysis of the Synechocystis isolates for slr1736 and sll1899showed complete segregation of the mutant genome, meaning no copies ofthe wild type genome could be detected in these strains. This suggeststhat function of the native gene is not essential for cell function.HPLC analysis of these same isolates showed that the sll1899 strain hadno detectable reduction in tocopherol levels. However, the straincarrying the knockout for slr1736 produced no detectable levels oftocopherol.

The amino acid sequences for the Synechocystis knockouts are comparedusing ClustalW, and are provided in Table 3 below. Provided are thepercent identities, percent similarity, and the percent gap. Thealignment of the sequences is provided in FIG. 21.

TABLE 3 Slr1736 slr0926 sll1899 slr0056 slr1518 slr1736 % identity 14 1218 11 % similar 29 30 34 26 % gap 8 7 10 5 slr0926 % identity 20 19 14 %similar 39 32 28 % gap 7 9 4 sll1899 % identity 17 13 % similar 29 29 %gap 12 9 slr0056 % identity 15 % similar 31 % gap 8 slr1518 % identity %similar % gap

Amino acid sequence comparisons are performed using various Arabidopsisprenyltransferase sequences and the Synechocystis sequences. Thecomparisons are presented in Table 4 below. Provided are the percentidentities, percent similarity, and the percent gap. The alignment ofthe sequences is provided in FIG. 22.

TABLE 4 ATPT2 slr1736 ATPT3 slr0926 ATPT4 sll1899 ATPT12 slr0056 ATPT8slr1518 ATPT2 29 9 9 8 8 12 9 7 9 46 23 21 20 20 28 23 21 20 27 13 28 2329 11 24 25 24 slr1736 9 13 8 12 13 15 8 10 19 28 19 28 26 33 21 26 3412 34 15 26 10 12 10 ATPT3 23 11 14 13 10 5 11 36 26 26 26 21 14 22 2921 31 16 30 30 30 12 20 17 20 11 14 slr0926 24 37 28 33 24 29 33 12 2510 11 9 18 11 8 6 7 ATPT4 33 23 18 16 19 28 19 32 32 33 13 17 10 12sll1899 24 30 23 26 27 13 10 11 52 8 11 ATPT12 66 19 26 18 25 23 9 13slr0056 23 32 10 8 7 ATPT8 23 7 slr1518

4C. Phytyl Prenyltransferase Enzyme Assays

[³H] Homogentisic acid in 0.1% H₃PO₄ (specific radioactivity 40Ci/mmol). Phytyl pyrophosphate was synthesized as described by Joo, etal. (1973) Can J. Biochem. 51:1527. 2-methyl-6-phytylquinol and2,3-dimethyl-5-phytylquinol were synthesized as described by Soll, etal. (1980) Phytochemistry 19:215. Homogentisic acid, α, β, δ, andγ-tocopherol, and tocol, were purchased commercially.

The wild-type strain of Synechocystis sp. PCC 6803 was grown in BG11medium with bubbling air at 30° C. under 50 μE.m⁻².s⁻¹ fluorescentlight, and 70% relative humidity. The growth medium of slr1736 knock-out(potential PPT) strain of this organism was supplemented with 25 μg mL⁻¹kanamycin. Cells were collected from 0.25 to 1 liter culture bycentrifugation at 5000 g for 10 min and stored at −80° C.

Total membranes were isolated according to Zak's procedures with somemodifications (Zak, et al. (1999) Eur J. Biochem 261:311). Cells werebroken on a French press. Before the French press treatment, the cellswere incubated for 1 hour with lysozyme (0.5%, w/v) at 30° C. in amedium containing 7 mM EDTA, 5 mM NaCl and 10 mM Hepes-NaOH, pH 7.4. Thespheroplasts were collected by centrifugation at 5000 g for 10 min andresuspended at 0.1-0.5 mg chlorophyll·mL⁻¹ in 20 mM potassium phosphatebuffer, pH 7.8. Proper amount of protease inhibitor cocktail and DNAaseI from Boehringer Mannheim were added to the solution. French presstreatments were performed two to three times at 100 MPa. After breakage,the cell suspension was centrifuged for 10 min at 5000 g to pelletunbroken cells, and this was followed by centrifugation at 100 000 g for1 hour to collect total membranes. The final pellet was resuspended in abuffer containing 50 mM Tris-HCL and 4 mM MgCl₂.

Chloroplast pellets were isolated from 250 g of spinach leaves obtainedfrom local markets. Devined leaf sections were cut into grinding buffer(2 l/250 g leaves) containing 2 mM EDTA, 1 mM MgCl₂, 1 mM MnCl₂, 0.33 Msorbitol, 0.1% ascorbic acid, and 50 mM Hepes at pH 7.5. The leaves werehomogenized for 3 sec three times in a 1-L blendor, and filtered through4 layers of mirocloth. The supernatant was then centrifuged at 5000 gfor 6 min. The chloroplast pellets were resuspended in small amount ofgrinding buffer (Douce, et al Methods in Chloroplast Molecular Biology,239 (1982)

Chloroplasts in pellets can be broken in three ways. Chloroplast pelletswere first aliquoted in 1 mg of chlorophyll per tube, centrifuged at6000 rpm for 2 min in microcentrifuge, and grinding buffer was removed.Two hundred microliters of Triton X-100 buffer (0.1% Triton X-100, 50 mMTris-HCl pH 7.6 and 4 mM MgCl₂) or swelling buffer (10 mM Tris pH 7.6and 4 mM MgCl₂) was added to each tube and incubated for ½ hour at 4° C.Then the broken chloroplast pellets were used for the assay immediately.In addition, broken chloroplasts can also be obtained by freezing inliquid nitrogen and stored at −80° C. for ½ hour, then used for theassay.

In some cases chloroplast pellets were further purified with 40%/80%percoll gradient to obtain intact chloroplasts. The intact chloroplastswere broken with swelling buffer, then either used for assay or furtherpurified for envelope membranes with 20.5%/31.8% sucrose densitygradient (Sol, et al (1980) supra). The membrane fractions werecentrifuged at 100 000 g for 40 min and resuspended in 50 mM Tris-HCl pH7.6, 4 mM MgCl₂.

Various amounts of [³H]HGA, 40 to 60 μM unlabelled HGA with specificactivity in the range of 0.16 to 4 Ci/mmole were mixed with a properamount of 1M Tris-NaOH pH 10 to adjust pH to 7.6. HGA was reduced for 4min with a trace amount of solid NaBH₄. In addition to HGA, standardincubation mixture (final vol 1 mL) contained 50 mM Tris-HCl, pH 7.6,3-5 mM MgCl₂, and 100 μM phytyl pyrophosphate. The reaction wasinitiated by addition of Synechocystis total membranes, spinachchloroplast pellets, spinach broken chloroplasts, or spinach envelopemembranes. The enzyme reaction was carried out for 2 hour at 23° C. or30° C. in the dark or light. The reaction is stopped by freezing withliquid nitrogen, and stored at −80° C. or directly by extraction.

A constant amount of tocol was added to each assay mixture and reactionproducts were extracted with a 2 mL mixture of chloroform/methanol (1:2,v/v) to give a monophasic solution. NaCl solution (2 mL; 0.9%) was addedwith vigorous shaking. This extraction procedure was repeated threetimes. The organic layer containing the prenylquinones was filteredthrough a 20 mμ filter, evaporated under N₂, and then resuspended in 100μL of ethanol.

The samples were mainly analyzed by Normal-Phase HPLC method (Isocratic90% Hexane and 10% Methyl-t-butyl ether), and use a Zorbax silicacolumn, 4.6×250 mm. The samples were also analyzed by Reversed-PhaseHPLC method (Isocratic 0.1% H₃PO₄ in MeOH), and use a Vydac 201HS54 C18column; 4.6×250 mm coupled with an All-tech C18 guard column. The amountof products were calculated based on the substrate specificradioactivity, and adjusted according to the % recovery based on theamount of internal standard.

The amount of chlorophyll was determined as described in Arnon (1949)Plant Physiol. 24:1. Amount of protein was determined by the Bradfordmethod using gamma globulin as a standard (Bradford, (1976) Anal.Biochem. 72:248)

Results of the assay demonstrate that 2-Methyl-6-Phytylplastoquinone isnot produced in the Synechocystis slr1736 knockout preparations. Theresults of the phytyl prenyltransferase enzyme activity assay for theslr1736 knock out are presented in FIG. 23.

4D. Complementation of the slr1736 knockout with ATPT2

In order to determine whether ATPT2 could complement the knockout ofslr1736 in Synechocystis 6803, a plasmid was constructed to express theATPT2 sequence from the TAC promoter. A vector, plasmid psl1211, wasobtained from the lab of Dr. Himadri Pakrasi of Washington University,and is based on the plasmid RSF1010 which is a broad host range plasmid(Ng W.-O., Zentella R., Wang, Y., Taylor J-S. A., Pakrasi, H. B. 2000.phrA, the major photoreactivating factor in the cyanobacteriumSynechocystis sp. strain PCC 6803 codes for a cyclobutane pyrimidinedimer specific DNA photolyase. Arch. Microbiol. (in press)). The ATPT2gene was isolated from the vector pCGN10817 by PCR using the followingprimers. ATPT2nco.pr 5′-CCATGGATTCGAGTAAAGTTGTCGC (SEQ ID NO:89);ATPT2ri.pr-5′-GAATTCACTTCAAAAAAGGTAACAG (SEQ ID NO:90). These primerswill remove approximately 112 BP from the 5′ end of the ATPT2 sequence,which is thought to be the chloroplast transit peptide. These primerswill also add an NcoI site at the 5′ end and an EcoRI site at the 3′ endwhich can be used for sub-cloning into subsequent vectors. The PCRproduct from using these primers and pCGN10817 was ligated into pGEM Teasy and the resulting vector pMON21689 was confirmed by sequencingusing the m13forward and m13reverse primers. The NcoI/EcoRI fragmentfrom pMON21689 was then ligated with the EagI/EcoRI and EagI/NcoIfragments from psl1211 resulting in pMON21690. The plasmid pMON21690 wasintroduced into the slr1736 Synechocystis 6803 KO strain viaconjugation. Cells of s1906 (a helper strain) and DH10B cells containingpMON21690 were grown to log phase (O.D. 600=0.4) and 1 ml was harvestedby centrifugation. The cell pellets were washed twice with a sterileBG-11 solution and resuspended in 200 ul of BG-11. The following wasmixed in a sterile eppendorf tube: 50 ul SL906, 50 ul DH10B cellscontaining pMON21690, and 100 ul of a fresh culture of the slr1736Synechocystis 6803 KO strain (O.D. 730=0.2-0.4). The cell mixture wasimmediately transferred to a nitrocellulose filter resting on BG-11 andincubated for 24 hours at 30 C and 2500 LUX (50 ue) of light. The filterwas then transferred to BG-11 supplemented with 10 ug/ml Gentamycin andincubated as above for ˜5 days. When colonies appeared, they were pickedand grown up in liquid BG-11+Gentamycin 10 ug/ml. (Elhai, J. and Wolk,P. 1988. Conjugal transfer of DNA to Cyanobacteria. Methods inEnzymology 167, 747-54) The liquid cultures were then assayed fortocopherols by harvesting 1 ml of culture by centrifugation, extractingwith ethanol/pyrogallol, and HPLC separation. The slr1736 Synechocystis6803 KO strain, did not contain any detectable tocopherols, while theslr1736 Synechocystis 6803 KO strain transformed with pmon21690contained detectable alpha tocopherol. A Synechocystis 6803 straintransformed with psl1211 (vector control) produced alpha tocopherol aswell.

4E: Additional Evidence of Prenyltransferase Activity

To test the hypothesis that slr736 or ATPT2 are sufficient as singlegenes to obtain phytyl prenyltransferase activity, both genes wereexpressed in SF9 cells and in yeast. When either slr1736 or ATPT2 wereexpressed in insect cells (Table 5) or in yeast, phytylprenyltransferase activity was detectable in membrane preparations,whereas membrane preparations of the yeast vector control, or membranepreparations of insect cells did not exhibit phytyl prenyltransferaseactivity.

TABLE 5 Phytyl prenyltransferase activity Enzyme activity Enzyme source[pmol/mg × h] slr1736 expressed in SF9 cells 20 ATPT2 expressed in SF9cells 6 SF9 cell control <0.05 Synechocystis 6803 0.25 Spinachchloroplasts 0.20

Example 5 Transgenic Plant Analysis 5A. Arabidopsis

Arabidopsis plants transformed with constructs for the sense orantisense expression of the ATPT proteins were analyzed by High PressureLiquid Chromatography (HPLC) for altered levels of total tocopherols, aswell as altered levels of specific tocopherols (alpha, beta, gamma, anddelta tocopherol).

Extracts of leaves and seeds were prepared for HPLC as follows. For seedextracts, 10 mg of seed was added to 1 g of microbeads (Biospec) in asterile microfuge tube to which 500 ul 1% pyrogallol (SigmaChem)/ethanol was added. The mixture was shaken for 3 minutes in a miniBeadbeater (Biospec) on “fast” speed. The extract was filtered through a0.2 um filter into an autosampler tube. The filtered extracts were thenused in HPLC analysis described below.

Leaf extracts were prepared by mixing 30-50 mg of leaf tissue with 1 gmicrobeads and freezing in liquid nitrogen until extraction. Forextraction, 500 ul 1% pyrogallol in ethanol was added to the leaf/beadmixture and shaken for 1 minute on a Beadbeater (Biospec) on “fast”speed. The resulting mixture was centrifuged for 4 minutes at 14,000 rpmand filtered as described above prior to HPLC analysis.

HPLC was performed on a Zorbax silica HPLC column (4.6 mm×250 mm) with afluorescent detection, an excitation at 290 nm, an emission at 336 nm,and bandpass and slits. Solvent A was hexane and solvent B wasmethyl-t-butyl ether. The injection volume was 20 ul, the flow rate was1.5 ml/min, the run time was 12 min (40° C.) using the gradient (Table6):

TABLE 6 Time Solvent A Solvent B  0 min. 90% 10% 10 min. 90% 10% 11 min.25% 75% 12 min. 90% 10%

Tocopherol standards in 1% pyrogallol/ethanol were also run forcomparison (alpha tocopherol, gamma tocopherol, beta tocopherol, deltatocopherol, and tocopherol (tocol) (all from Matreya).

Standard curves for alpha, beta, delta, and gamma tocopherol werecalculated using Chemstation software. The absolute amount of componentx is: Absolute amount of x=Response×RF_(x)×dilution factor whereResponse is the area of peak x, RF_(x) is the response factor forcomponent x (Amount_(x)/Response_(x)) and the dilution factor is 500 ul.The ng/mg tissue is found by: total ng component/mg plant tissue.

Results of the HPLC analysis of seed extracts of transgenic Arabidopsislines containing pMON10822 for the expression of ATPT2 from the napinpromoter are provided in FIG. 24.

HPLC analysis results of segregating T2 Arabidopsis seed tissueexpressing the ATPT2 sequence from the napin promoter (pCGN10822)demonstrates an increased level of tocopherols in the seed. Totaltocopherol levels are increased as much as 50% over the total tocopherollevels of non-transformed (wild-type) Arabidopsis plants (FIG. 25).Homozygous progeny from the top 3 lines (T3 seed) have up to a two-fold(100%) increase in total tocopherol levels over control Arabidopsis seed(FIG. 26.)

Furthermore, increases of particular tocopherols are also increased intransgenic Arabidopsis plants expressing the ATPT2 nucleic acid sequencefrom the napin promoter. Levels of delta tocopherol in these lines areincreased greater than 3 fold over the delta tocopherol levels obtainedfrom the seeds of wild type Arabidopsis lines. Levels of gammatocopherol in transgenic Arabidopsis lines expressing the ATPT2 nucleicacid sequence are increased as much as about 60% over the levelsobtained in the seeds of non-transgenic control lines. Furthermore,levels of alpha tocopherol are increased as much as 3 fold over thoseobtained from non-transgenic control lines.

Results of the HPLC analysis of seed extracts of transgenic Arabidopsislines containing pCGN10803 for the expression of ATPT2 from the enhanced³⁵S promoter (antisense orientation) are provided in FIG. 25. Two lineswere identified that have reduced total tocopherols, up to a ten-folddecrease observed in T3 seed compared to control Arabidopsis (FIG. 27.)

5B. Canola

Brassica napus, variety SP30021, was transformed with pCGN10822(napin-ATPT2-napin 3′, sense orientation) using Agrobacteriumtumefaciens-mediated transformation. Flowers of the R0 plants weretagged upon pollination and developing seed was collected at 35 and 45days after pollination (DAP).

Developing seed was assayed for tocopherol levels, as described abovefor Arabidopsis. Line 10822-1 shows a 20% increase of total tocopherols,compared to the wild-type control, at 45 DAP. FIG. 28 shows totaltocopherol levels measured in developing canola seed.

Example 6 Sequences to Tocopherol Cyclase

6A. Preparation of the slr1737 Knockout

The Synechocystis sp. 6803 slr1737 knockout was constructed by thefollowing method. The GPS™-1 Genome Priming System (New England Biolabs)was used to insert, by a Tn7 Transposase system, a Kanamycin resistancecassette into slr1737. A plasmid from a Synechocystis genomic libraryclone containing 652 base pairs of the targeted orf (Synechcocystisgenome base pairs 1324051-1324703; the predicted orf base pairs1323672-1324763, as annotated by Cyanobase) was used as target DNA. Thereaction was performed according to the manufacturers protocol. Thereaction mixture was then transformed into E. coli DH10Belectrocompetant cells and plated. Colonies from this transformationwere then screened for transposon insertions into the target sequence byamplifying with M13 Forward and Reverse

Universal primers, yielding a product of 652 base pairs plus ˜1700 basepairs, the size of the transposon kanamycin cassette, for a totalfragment size of ˜2300 base pairs. After this determination, it was thennecessary to determine the approximate location of the insertion withinthe targeted orf, as 100 base pairs of orf sequence was estimated asnecessary for efficient homologous recombination in Synechocystis. Thiswas accomplished through amplification reactions using either of theprimers to the ends of the transposon, Primer S (5′ end) or N (3′ end),in combination with either a M13 Forward or Reverse primer. That is,four different primer combinations were used to map each potentialknockout construct: Primer S-M13 Forward, Primer S-M13 Reverse, PrimerN-M13 Forward, Primer N-M13 Reverse. The construct used to transformSynechocystis and knockout slr1737 was determined to consist of aapproximately 150 base pairs of slr1737 sequence on the 5′ side of thetransposon insertion and approximately 500 base pairs on the 3′ side,with the transcription of the orf and kanamycin cassette in the samedirection. The nucleic acid sequence of slr1737 is provided in SEQ IDNO:38 the deduced amino acid sequence is provided in SEQ ID NO:39.

Cells of Synechocystis 6803 were grown to a density of ˜2×10⁸ cells perml and harvested by centrifugation. The cell pellet was re-suspended infresh BG-11 medium at a density of 1×10⁹ cells per ml and usedimmediately for transformation. 100 ul of these cells were mixed with 5ul of mini prep DNA and incubated with light at 30 C for 4 hours. Thismixture was then plated onto nylon filters resting on BG-11 agarsupplemented with TES ph8 and allowed to grow for 12-18 hours. Thefilters were then transferred to BG-11 agar+TES+5 ug/ml kanamycin andallowed to grow until colonies appeared within 7-10 days (Packer andGlazer, 1988). Colonies were then picked into BG-11 liquid mediacontaining 5 ug/ml kanamycin and allowed to grow for 5 days. These cellswere then transferred to Bg-11 media containing 10 ug/ml kanamycin andallowed to grow for 5 days and then transferred to Bg-11+kanamycin at 25ug/ml and allowed to grow for 5 days. Cells were then harvested for PCRanalysis to determine the presence of a disrupted ORF and also for HPLCanalysis to determine if the disruption had any effect on tocopherollevels.

PCR analysis of the Synechocystis isolates, using primers to the ends ofthe slr1737 orf, showed complete segregation of the mutant genome,meaning no copies of the wild type genome could be detected in thesestrains. This suggests that function of the native gene is not essentialfor cell function. HPLC analysis of the strain carrying the knockout forslr1737 produced no detectable levels of tocopherol.

6B. The Relation of slr1737 and slr1736

The slr1737 gene occurs in Synechocystis downstream and in the sameorientation as slr1736, the phytyl prenyltransferase. In bacteria thisproximity often indicates an operon structure and therefore anexpression pattern that is linked in all genes belonging to this operon.Occasionally such operons contain several genes that are required toconstitute one enzyme. To confirm that slr1737 is not required forphytyl prenyltransferase activity, phytyl prenyltransferase was measuredin extracts from the Synechocystis slr1737 knockout mutant. FIG. 29shows that extracts from the Synechocystis slr1737 knockout mutant stillcontain phytyl prenyltransferase activity. The molecular organization ofgenes in Synechocystis 6803 is shown in A. Figures B and C show HPLCtraces (normal phase HPLC) of reaction products obtained with membranepreparations from Synechocystis wild type and slr1737 membranepreparations, respectively.

The fact that slr1737 is not required for the PPT activity providesadditional data that ATPT2 and slr1736 encode phytyl prenyltransferases.

6C Synechocystis Knockouts

Synechocystis 6803 wild type and Synechocystis slr1737 knockout mutantwere grown photoautotrophically. Cells from a 20 ml culture of the latelogarithmic growth phase were harvested and extracted with ethanol.Extracts were separated by isocratic normal-phase HPLC using aHexane/Methyl-t-butyl ether (95/5) and a Zorbax silica column, 4.6×250mm. Tocopherols and tocopherol intermediates were detected byfluorescence (excitement 290 nm, emission 336 nm) (FIG. 30).

Extracts of Synechocystis 6803 contained a clear signal ofalpha-tocopherol. 2,3-Dimethyl-5-phytylplastoquinol was below the limitof detection in extracts from the Synechocystis wild type (C). Incontrast, extracts from the Synechocystis slr1737 knockout mutant didnot contain alpha-tocopherol, but contained2,3-dimethyl-5-phytylplastoquinol (D), indicating that the interruptionof slr1737 has resulted in a block of the2,3-dimethyl-5-phytylplastoquinol cyclase reaction.

Chromatograms of standard compounds alpha, beta, gamma, delta-tocopheroland 2,3-dimethyl-5-phytylplastoquinol are shown in A and B.Chromatograms of extracts form Synechocystis wild type and theSynechocystis slr1737 knockout mutant are shown in C and D,respectively. Abbreviations: 2,3-DMPQ,2,3-dimethyl-5-phytylplastoquinol.

6D. Incubation with Lysozyme Treated Synechocystis

Synechocystis 6803 wild type and slr1737 knockout mutant cells from thelate logarithmic growth phase (approximately 1 g wet cells perexperiment in a total volume of 3 ml) were treated with Lysozyme andsubsequently incubated with S-adenosylmethionine, andphytylpyrophosphate, plus radiolabelled homogentisic acid. After 17 hincubation in the dark at room temperature the samples were extractedwith 6 ml chloroform/methanol (1/2 v/v). Phase separation was obtainedby the addition of 6 ml 0.9% NaCl solution. This procedure was repeatedthree times. Under these conditions 2,3-dimethyl-5-phytylplastoquinol isoxidized to form 2,3-dimethyl-5-phytylplastoquinone.

The extracts were analyzed by normal phase and reverse phase HPLC. Usingextracts from wild type Synechocystis cells radiolabelledgamma-tocopherol and traces of radiolabelled2,3-dimethyl-5-phytylplastoquinone were detected. When extracts from theslr1737 knockout mutant were analyzed, only radiolabelled2,3-dimethyl-5-phytylplastoquinone was detectable. The amount of2,3-dimethyl-5-phytylplastoquinone was significantly increased comparedto wild type extracts. Heat treated samples of the wild type and theslr1737 knockout mutant did not produce radiolabelled2,3-dimethyl-5-phytylplastoquinone, nor radiolabelled tocopherols. Theseresults further support the role of the slr1737 expression product inthe cyclization of 2,3-dimethyl-5-phytylplastoquinol.

6E. Arabidopsis Homologue to slr1737

An Arabidopsis homologue to slr1737 was identified from a BLASTALLsearch using Synechocystis sp 6803 gene slr1737 as the query, in bothpublic and proprietary databases. SEQ ID NO:109 and SEQ ID NO:110 arethe DNA and translated amino acid sequences, respectively, of theArabidopsis homologue to slr1737. The start if found at the ATG at base56 in SEQ ID NO:109.

The sequences obtained for the homologue from the proprietary databasediffers from the public database (F4D11.30, BAC AL022537), in having astart site 471 base pairs upstream of the start identified in the publicsequence. A comparison of the public and proprietary sequences isprovided in FIG. 31. The correct start correlates within the publicdatabase sequence is at 12080, while the public sequence start is givenas being at 11609.

Attempts to amplify a slr1737 homologue were unsuccessful using primersdesigned from the public database, while amplification of the gene wasaccomplished with primers obtained from SEQ ID NO:109.

Analysis of the protein sequence to identify transit peptide sequencepredicted two potential cleavage sites, one between amino acids 48 and49, and the other between amino acids 98 and 99.

6F. slr1737 Protein Information

The slr1737 orf comprises 363 amino acid residues and has a predicted MWof 41 kDa (SEQ ID NO: 39). Hydropathic analysis indicates the protein ishydrophillic (FIG. 32).

The Arabidopsis homologue to slr1737 (SEQ ID xx) comprises 488 aminoacid residues, has a predicted MW of 55 kDa, and a has a putativetransit peptide sequence comprising the first 98 amino acids. Thepredicted MW of the mature form of the Arabidopsis homologue is 44 kDa.The hydropathic plot for the Arabidopsis homologue also reveals that itis hydrophillic (FIG. 33). Further blast analysis of the Arabidopsishomologue reveals limited sequence identity (25% sequence identity) withthe beta-subunit of respiratory nitrate reductase. Based on the sequenceidentity to nitrate reductase, it suggests the slr1737 orf is an enzymethat likely involves general acid catalysis mechanism.

Investigation of known enzymes involved in tocopherol metabolismindicated that the best candidate corresponding to the general acidmechanism is the tocopherol cyclase. There are many known examples ofcyclases including, tocopherol cyclase, chalcone isomerase, lycopenecyclase, and aristolochene synthase. By further examination of themicroscopic catalytic mechanism of phytoplastoquinol cyclization, as anexample, chalcone isomerase has a catalytic mechanism most similar totocopherol cyclase. (FIG. 34).

Multiple sequence alignment was performed between slr1737, slr1737Arabidopsis homologue and the Arabidopsis chalcone isomerase(Genbank:P41088) (FIG. 35). 65% of the conserved residues among thethree enzymes are strictly conserved within the known chalconeisomerases. The crystal structure of alfalfa chalcone isomerase has beensolved (Jez, Joseph M., Bowman, Marianne E., Dixon, Richard A., andNoel, Joseph P. (2000) “Structure and mechanism of the evolutionarilyunique plant enzyme chalcone isomerase”. Nature Structural Biology 7:786-791.) It has been demonstrated tyrosine (Y) 106 of the alfalfachalcone isomerase serves as the general acid during cyclizationreaction (Genbank: P28012). The equivalent residue in slr1737 and theslr1737 Arabidopsis homolog is lysine (K), which is an excellentcatalytic residue as general acid.

The information available from partial purification of tocopherolcyclase from Chlorella protothecoides (U.S. Pat. No. 5,432,069), i.e.,described as being glycine rich, water soluble and with a predicted MWof 48-50 kDa, is consistent with the protein informatics informationobtained for the slr1737 and the Arabidopsis slr1737 homologue.

All publications and patent applications mentioned in this specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claim.

1-25. (canceled)
 26. A method for the alteration of the isoprenoidcontent in a host cell, said method comprising; expressing in a hostcell a polynucleotide molecule comprising a sequence encoding atranscriptional initiation region functional in the host cell operablylinked to a nucleic acid sequence encoding a tocopherol cyclase,selected from the group consisting of: a) a polynucleotide sequenceencoding the polypeptide of SEQ ID NO: 39; b) a polynucleotide sequencethat has at least 70% identity to SEQ ID NO: 38; c) a polynucleotidesequence encoding the polypeptide that has at least 70% identity to SEQID NO: 39; and d) a polynucleotide sequence that hybridizes, understringent conditions, to SEQ ID NO: 38, wherein said isoprenoid compoundselected from the group of tocopherols and tocotrienols.
 27. The methodaccording to claim 26, wherein said host cell is selected from the groupconsisting of a prokaryotic cell and a eukaryotic cell.
 28. The methodaccording to claim 27, wherein said prokaryotic cell is a Synechocystissp.
 29. The method according to claim 27, wherein said eukaryotic cellis a plant cell.
 30. The method according to claim 29, wherein saidplant cell is selected from the group consisting of an Arabidopsis,soybean, corn, rice, wheat, leek canola, leek, cotton, and tomato plantcell.
 31. A method for producing an isoprenoid compound of interest in ahost cell, said method comprising obtaining a transformed host cell,said host cell comprising in its genome: a polynucleotide constructcomprising nucleic acid sequence encoding a tocopherol cyclase selectedfrom the group consisting of: a) a polynucleotide sequence encoding thepolypeptide of SEQ ID NO: 39; b) a polynucleotide sequence that has atleast 70% identity to SEQ ID NO: 38; c) a polynucleotide sequenceencoding the polypeptide that has at least 70% identity to SEQ ID NO:39; and d) a polynucleotide sequence that hybridizes, under stringentconditions, to SEQ ID NO: 38, operably linked to a transcriptionalinitiation region functional in the host cell, wherein said isoprenoidcompound selected from the group of tocopherols and tocotrienols. 32.The method according to claim 31, wherein said host cell is selectedfrom the group consisting of a prokaryotic cell and a eukaryotic cell.33. The method according to claim 32, wherein said prokaryotic cell is aSynechocystis sp.
 34. The method according to claim 32, wherein saideukaryotic cell is a plant cell.
 35. The method according to claim 34,wherein said plant cell is selected from the group consisting of anArabidopsis, soybean, corn, rice, wheat, leek canola, leek, cotton, andtomato plant cell.
 36. A method for increasing the biosynthetic flux ina host cell toward production of an isoprenoid compound, said methodcomprising; expressing in a host cell a polynucleotide constructcomprising as operably linked components, a transcriptional initiationregion functional in the host cell and a DNA encoding a tocopherolcyclase selected from the group consisting of: a) a polynucleotidesequence encoding the polypeptide of SEQ ID NO: 39; b) a polynucleotidesequence that has at least 70% identity to SEQ ID NO: 38; c) apolynucleotide sequence encoding the polypeptide that has at least 70%identity to SEQ ID NO: 39; and d) a polynucleotide sequence thathybridizes, under stringent conditions, to SEQ ID NO: 38, wherein saidisoprenoid compound selected from the group of tocopherols andtocotrienols.
 37. The method according to claim 36, wherein said hostcell is selected from the group consisting of a prokaryotic cell and aeukaryotic cell.
 38. The method according to claim 37, wherein saidprokaryotic cell is a Synechocystis sp.
 39. The method according toclaim 37, wherein said eukaryotic cell is a plant cell.
 40. The methodaccording to claim 39, wherein said plant cell is obtained from a plantselected from the group consisting an Arabidopsis, soybean, corn, rice,wheat, leek canola, leek, cotton, and tomato plant cell.
 41. The methodaccording to claim 39, wherein said transcriptional initiation region isa seed-specific promoter.
 42. The method of claim 29, wherein the plantcell is comprised in a plant.
 43. The method of claim 34, wherein theplant cell is comprised in a plant.
 44. The method of claim 39, whereinthe plant cell is comprised in a plant.